Chlamydia antigens and corresponding DNA fragments and uses thereof

ABSTRACT

The present invention provides a method of nucleic acid, including DNA, immunization of a host, including humans, against disease caused by infection by a strain of Chlamydia, specifically  C. pneumoniae,  employing a vector containing a nucleotide sequence encoding a 98 kDa outer membrane protein of a strain of  Chlamydia pneumoniae  and a promoter to effect expression of the 98 kDa outer membrane protein gene in the host. Modifications are possible within the scope of this invention.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 60/113,439, filed Dec. 1, 1998 and U.S. Provisional Application No.60/132,272, filed May 3, 1999.

FIELD OF INVENTION

[0002] The present invention relates to the Chlamydia 98KDa outer membrane protein antigen and corresponding DNA molecules, which can be used to prevent and treat Chlamydia infection in mammals, such as humans.

BACKGROUND OF THE INVENTION

[0003] Chlamydiae are prokaryotes. They exhibit morphologic and structural similarities to gram-negative bacteria including a trilaminar outer membrane, which contains lipopolysaccharide and several membrane proteins that are structurally and functionally analogous to proteins found in E coli. They are obligate intra-cellular parasites with a unique biphasic life cycle consisting of a metabolically inactive but infectious extracellular stage and a replicating but non-infectious intracellular stage. The replicative stage of the life-cycle takes place within a membrane-bound inclusion which sequesters the bacteria away from the cytoplasm of the infected host cell.

[0004]C. pneumoniae is a common human pathogen, originally described as the TWAR strain of Chlamydia psittaci but subsequently recognised to be a new species. C. pneumoniae is antigenically, genetically and morphologically distinct from other chlamydia species (C. trachomatis, C. pecorum and C. psittaci). It shows 10% or less DNA sequence homology with either of C. trachomatis or C.psittaci.

[0005]C. pneumoniae is a common cause of community acquired pneumonia, only less frequent than Streptococcus pneumoniae and Mycoplasma pneumoniae (Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Campos et al. (1995) Investigation of Ophthalmology and Visual Science 36:1477). It can also cause upper respiratory tract symptoms and disease, including bronchitis and sinusitis (Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Grayston et al (1990) Journal of Infectious Diseases 161:618; Marrie (1993) Clinical Infectious Diseases. 18:501; Wang et al (1986) Chlamydial infections Cambridge University Press, Cambridge. p. 329. The great majority of the adult population (over 60%) has antibodies to C. pneumoniae (Wang et al (1986) Chlamydial infections. Cambridge University Press, Cambridge. p. 329), indicating past infection which was unrecognized or asymptomatic.

[0006]C. pneumoniae infection usually presents as an acute respiratory disease (i.e., cough, sore throat, hoarseness, and fever; abnormal chest sounds on auscultation). For most patients, the cough persists for 2 to 6 weeks, and recovery is slow. In approximately 10% of these cases, upper respiratory tract infection is followed by bronchitis or pneumonia. Furthermore, during a C. pneumoniae epidemic, subsequent co-infection with pneumococcus has been noted in about half of these pneumonia patients, particularly in the infirm and the elderly. As noted above, there is more and more evidence that C. pneumoniae infection is also linked to diseases other than respiratory infections.

[0007] The reservoir for the organism is presumably people. In contrast to C. psittaci infections, there is no known bird or animal reservoir. Transmission has not been clearly defined. It may result from direct contact with secretions, from fomites, or from airborne spread. There is a long incubation period, which may last for many months. Based on analysis of epidemics, C. pneumoniae appears to spread slowly through a population (case-to-case interval averaging 30 days) because infected persons are inefficient transmitters of the organism. Susceptibility to C. pneumoniae is universal. Reinfections occur during adulthood, following the primary infection as a child. C. pneumoniae appears to be an endemic disease throughout the world, noteworthy for superimposed intervals of increased incidence (epidemics) that persist for 2 to 3 years. C. trachomatis infection does not confer cross-immunity to C. pneumoniae. Infections are easily treated with oral antibiotics, tetracycline or erythromycin (2 g/d, for at least 10 to 14 d). A recently developed drug, azithromycin, is highly effective as a single-dose therapy against chlamydial infections.

[0008] In most instances, C. pneumoniae infection is often mild and without complications, and up to 90% of infections are subacute or unrecognized. Among children in industrialized countries, infections have been thought to be rare up to the age of 5 y, although a recent study (E Normann et al, Chlamydia pneumoniae in children with acute respiratory tract infections, Acta Paediatrica, 1998, Vol 87, Iss 1, pp 23-27) has reported that many children in this age group show PCR evidence of infection despite being seronegative, and estimates a prevalence of 17-19% in 2-4 y olds. In developing countries, the seroprevalence of C. pneumoniae antibodies among young children is elevated, and there are suspicions that C. pneumoniae may be an important cause of acute lower respiratory tract disease and mortality for infants and children in tropical regions of the world.

[0009] From seroprevalence studies and studies of local epidemics, the initial C. pneumoniae infection usually happens between the ages of 5 and 20 y. In the USA, for example, there are estimated to be 30,000 cases of childhood pneumonia each year caused by C. pneumoniae. Infections may cluster among groups of children or young adults (e.g., school pupils or military conscripts).

[0010]C. pneumoniae causes 10 to 25% of community-acquired lower respiratory tract infections (as reported from Sweden, Italy, Finland, and the USA). During an epidemic, C. pneumonia infection may account for 50 to 60% of the cases of pneumonia. During these periods, also, more episodes of mixed infections with S. pneumoniae have been reported.

[0011] Reinfection during adulthood is common; the clinical presentation tends to be milder. Based on population seroprevalence studies, there tends to be increased exposure with age, which is particularly evident among men. Some investigators have speculated that a persistent, asymptomatic C. pneumoniae infection state is common.

[0012] In adults of middle age or older, C. pneumoniae infection may progress to chronic bronchitis and sinusitis. A study in the USA revealed that the incidence of pneumonia caused by C. pneumoniae in persons younger than 60 years is 1 case per 1,000 persons per year; but in the elderly, the disease incidence rose three-fold. C. pneumoniae infection rarely leads to hospitalization, except in patients with an underlying illness.

[0013] Of considerable importance is the association of atherosclerosis and C. pneumoniae infection. There are several epidemiological studies showing a correlation of previous infections with C. pneumoniae and heart attacks, coronary artery and carotid artery disease (Saikku et al. (1988) Lancet;ii:983; Thom et al. (1992) JAMA 268:68; Linnanmaki et al. (1993), Circulation 87:1030; Saikku et al. (1992)Annals Internal Medicine 116:273; Melnick et al (1993) American Journal of Medicine 95:499). Moreover, the organisms has been detected in atheromas and fatty streaks of the coronary, carotid, peripheral arteries and aorta (Shor et al. (1992) South African. Medical Journal 82:158; Kuo et al. (1993) Journal of Infectious Diseases 167:841; Kuo et al. (1993) Arteriosclerosis and Thrombosis 13:1500; Campbell et al (1995) Journal of Infectious Diseases 172:585; Chiu et al. Circulation, 1997 (In Press)). Viable C. pneumoniae has been recovered from the coronary and carotid artery (Ramirez et al (1996) Annals of Internal Medicine 125:979; Jackson et al. Abst. K121, p272, 36^(th) ICAAC, Sep. 15-18, 1996, New Orleans). Furthermore, it has been shown that C. pneumoniae can induce changes of atherosclerosis in a rabbit model (Fong et al (1997) Journal of Clinical Microbiolology 35:48). Taken together, these results indicate that it is highly probable that C. pneumoniae can cause atherosclerosis in humans, though the epidemiological importance of chlamydial atherosclerosis remains to be demonstrated.

[0014] A number of recent studies have also indicated an association between C. pneumoniae infection and asthma. Infection has been linked to wheezing, asthmatic bronchitis, adult-onset asthma and acute exacerbations of asthma in adults, and small-scale studies have shown that prolonged antibiotic treatment was effective at greatly reducing the severity of the disease in some individuals (Hahn D L, et al. Evidence for Chlamydia pneumoniae infection in steroid-dependent asthma. Ann Allergy Asthma Immunol. 1998 January; 80(1): 45-49.; Hahn D L, et al. Association of Chlamydia pneumoniae IgA antibodies with recently symptomatic asthma. Epidemiol Infect. 1996 December; 117(3): 513-517; Bjornsson E, et al. Serology of chlamydia in relation to asthma and bronchial hyperresponsiveness. Scand J Infect Dis. 1996; 28(1): 63-69.; Hahn D L. Treatment of Chlamydia pneumoniae infection in adult asthma: a before-after trial. J Fam Pract. 1995 October; 41(4): 345-351.; Allegra L, et al. Acute exacerbations of asthma in adults: role of Chlamydia pneumoniae infection. Eur Respir J. 1994 December; 7(12): 2165-2168.; Hahn D L, et al. Association of Chlamydia pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and adult-onset asthma. JAMA. Jul. 10, 1991; 266(2): 225-230).

[0015] In light of these results a protective vaccine against C. pneumoniae infection would be of considerable importance. There is not yet an effective vaccine for any human chlamydial infection. It is conceivable that an effective vaccine can be developed using physically or chemically inactivated Chlamydiae. However, such a vaccine does not have a high margin of safety. In general, safer vaccines are made by genetically manipulating the organism by attenuation or by recombinant means. Accordingly, a major obstacle in creating an effective and safe vaccine against human chlamydial infection has been the paucity of genetic information regarding Chlamydia, specifically C. pneumoniae.

[0016] Studies with C. trachomatis and C. psittaci indicate that safe and effective vaccine against Chlamydia is an attainable goal. For example, mice which have recovered from a lung infection with C. trachomatis are protected from infertility induced by a subsequent vaginal challenge (Pal et al. (1996) Infection and Immunity.64:5341). Similarly, sheep immunized with inactivated C. psittaci were protected from subsequent chlamydial-induced abortions and stillbirths (Jones et al. (1995) Vaccine 13:715). Protection from chlamydial infections has been associated with Th1 immune responses, particularly the induction of INFg-producing CD4+T-cells (Igietsemes et al. (1993) Immunology 5:317). The adoptive transfer of CD4+ cell lines or clones to nude or SCID mice conferred protection from challenge or cleared chronic disease (Igietseme et al (1993) Regional Immunology 5:317; Magee et al (1993) Regional Immunology 5: 305), and in vivo depletion of CD4+ T cells exacerbated disease post-challenge (Landers et al (1991) Infection & Immunity 59:3774; Magee et al (1995) Infection & Immunity 63:516). However, the presence of sufficiently high titres of neutralising antibody at mucosal surfaces can also exert a protective effect (Cotter et al. (1995) Infection and Immunity 63:4704).

[0017] Antigenic variation within the species C. pneumoniae. is not well documented due to insufficient genetic information, though variation is expected to exist based on C. trachomatis. Serovars of C. trachomatis are defined on the basis of antigenic variation in the major outer membrane protein (MOMP), but published C. pneumoniae MOMP gene sequences show no variation between several diverse isolates of the organism (Campbell et al (1990) Infection and Immunity 58:93; McCafferty et al (1995) Infection and Immunity 63:2387-9; Knudsen et al (19.96) Third Meeting of the European Society for Chlamydia Research, Vienna). The gene encoding a 76 kDa antigen has been cloned from a single strain of C. pneumoniae and the sequence published (Perez Melgosa et al., Infect. Immun. 1994. 62:880). An operon encoding the 9 kDa and 60 kDa cyteine-rich outer membrane protein genes has been described (Watson et al., Nucleic Acids Res (1990) 18:5299; Watson et al., Microbiology (1995) 141:2489). Many antigens recognized by immune sera to C. pneumoniae are conserved across all chlamydiae, but 98 kDa, 76 kDa and several other proteins may be C. pneumoniae-specific (Perez Melgosa et al., Infect. Immun. 1994. 62:880; Melgosa et al., FEMS Microbiol Lett (1993) 112 :199;, Campbell et al., J Clin Microbiol (1990) 28 :1261; Iijima et al., J Clin Microbiol (1994) 32:583). An assessment of the number and relative frequency of any C. pneumoniae serotypes, and the defining antigens, is not yet possible. The entire genome sequence of C. pneumoniae strain CWL-029 is now known (http://chlamydia-www.berkeley.edu:4231/) and as further sequences become available a better understanding of antigenic variation may be gained.

[0018] Many antigens recognised by immune sera to C. pneumoniae are conserved across all chlamydiae, but 98kDa, 76 kDa and 54 kDa proteins appear to be C. pneumoniae-specific (Campos et al. (1995) Investigation of Ophthalmology and Visual Science 36:1477; Marrie (1993) Clinical Infectious Diseases. 18:501; Wiedmann-Al-Ahmad M, et al. Reactions of polyclonal and neutralizing anti-p54 monoclonal antibodies with an isolated, species-specific 54-kilodalton protein of Chlamydia pneumoniae. Clin Diagn Lab Immunol. 1997 November; 4(6): 700-704). Immunoblotting of isolates with sera from patients does show variation of blotting patterns between isolates, indicating that serotypes C. pneumoniae may exist (Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Ramirez et al (1996) Annals of Internal Medicine 125:979). However, the results are potentially confounded by the infection status of the patients, since immunoblot profiles of a patient's sera change with time post-infection. An assessment of the number and relative frequency of any serotypes, and the defining antigens, is not yet possible.

[0019] Accordingly, a need exists for identifying and isolating polynucleotide sequences of C. pneumoniae for use in preventing and treating Chlamydia infection.

SUMMARY OF THE INVENTION

[0020] The present invention provides purified and isolated polynucleotide molecules that encode the Chlamydia 98KDa outer membrane protein which can be used in methods to prevent, treat, and diagnose Chlamydia infection. In one form of the invention, the polynucleotide molecules are DNA that encode polypeptides CPN100640 (SEQ ID Nos: 1 and 2).

[0021] Another form of the invention provides polypeptides corresponding to the isolated DNA molecules. The amino acid sequences of the corresponding encoded polypeptides are shown as SEQ ID No: 3 and 4, where SEQ ID No. 3 is the full length sequence and SEQ ID No. 4 is the post-translationally processed sequence.

[0022] Those skilled in the art will readily understand that the invention, having provided the polynucleotide sequences encoding the Chlamydia 98 KDa outer membrane protein, also provides polynucleotides encoding fragments derived from such polypeptides. Moreover, the invention is understood to provide mutants and derivatives of such polypeptides and fragments derived therefrom, which result from the addition, deletion, or substitution of non-essential amino acids as described herein. Those skilled in the art would also readily understand that the invention, having provided the polynucleotide sequences encoding Chlamydia polypeptides, further provides monospecific antibodies that specifically bind to such polypeptides.

[0023] The present invention has wide application and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention. Accordingly, the present invention further provides (i) a method for producing a polypeptide of the invention in a recombinant host system and related expression cassettes, vectors, and transformed or transfected cells; (ii) a vaccine, or a live vaccine vector such as a pox virus, Salmonella typhimurium, or Vibrio cholerae vector, containing a polynucleotide of the invention, such vaccines and vaccine vectors being useful for, e.g., preventing and treating Chlamydia infection, in combination with a diluent or carrier, and related pharmaceutical compositions and associated therapeutic and/or prophylactic methods; (iii) a therapeutic and/or prophylactic use of an RNA or DNA molecule of the invention, either in a naked form or formulated with a delivery vehicle, a polypeptide or combination of polypeptides, or a monospecific antibody of the invention, and related pharmaceutical compositions; (iv) a method for diagnosing the presence of Chlamydia in a biological sample, which can involve the use of a DNA or RNA molecule, a monospecific antibody, or a polypeptide of the invention; and (v) a method for purifying a polypeptide of the invention by antibody-based affinity chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further understood from the following description with reference to the drawings, in which:

[0025]FIG. 1 shows the nucleotide sequence of the CPN100640 (SEQ ID No: 1—entire sequence and SEQ ID No: 2—coding sequence) and the deduced amino acid sequence of the CPN100640 protein from Chlamydia pneumoniae (SEQ ID No: 3—full length and 4—processed). The sequence is encoded on the negative strand.

[0026]FIG. 2 shows the restriction enzyme analysis of the C. pneumoniae 98 kDa outer membrane protein gene.

[0027]FIG. 3 shows the construction and elements of plasmid pCAI640.

[0028]FIG. 4 illustrates protection against C. pneumoniae infection by pCAI640 following DNA immunization.

DETAILED DESCRIPTION OF INVENTION

[0029] An open reading frames (ORF) encoding the chlamydial 98KDa outer membrane protein has been identified from the C. pneumoniae genome. The gene encoding this protein has been inserted into an expression plasmid and shown to confer immune protection against chlamydial infection. Accordingly, this outer membrane protein and related polypeptides can be used to prevent and treat Chlamydia infection.

[0030] According to a first aspect of the invention, isolated polynucleotides are provided which encode the precursor and mature forms of Chlamydia polypeptides, whose amino acid sequences are SEQ ID Nos: 3 and 4 respectively.

[0031] The term “isolated polynucleotide” is defined as a polynucleotide removed from the environment in which it naturally occurs. For example, a naturally-occurring DNA molecule present in the genome of a living bacteria or as part of a gene bank is not isolated, but the same molecule separated from the remaining part of the bacterial genome, as a result of, e.g., a cloning event (amplification), is isolated. Typically, an isolated DNA molecule is free from DNA regions (e.g., coding regions) with which it is immediately contiguous at the 5′ or 3′ end, in the naturally occurring genome. Such isolated polynucleotides may be part of a vector or a composition and still be defined as isolated in that such a vector or composition is not part of the natural environment of such polynucleotide.

[0032] The polynucleotide of the invention is either RNA or DNA (cDNA, genomic DNA, or synthetic DNA), or modifications, variants, homologs or fragments thereof. The DNA is either double-stranded or single-stranded, and, if single-stranded, is either the coding strand or the non-coding (anti-sense) strand. Any one of the sequences that encode the polypeptides of the invention as shown in SEQ ID Nos: 1 and 2 is (a) a coding sequence, (b) a ribonucleotide sequence derived from transcription of (a), or (c) a coding sequence which uses the redundancy or degeneracy of the genetic code to encode the same polypeptides. By “polypeptide” or “protein” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application.

[0033] Consistent with the first aspect of the invention, amino acid sequences are provided which are homologous to either SEQ ID No: 3 or 4. As used herein, “homologous amino acid sequence” is any polypeptide which is encoded, in whole or in part, by a nucleic acid sequence which hybridizes at 25-35° C. below critical melting temperature (Tm), to any portion of the nucleic acid sequences of SEQ ID Nos: 1 and 2. A homologous amino acid sequence is one that differs from an amino acid sequence shown in SEQ ID No: 3 or 4 by one or more conservative amino acid substitutions. Such a sequence also encompass serotypic variants (defined below) as well as sequences containing deletions or insertions which retain inherent characteristics of the polypeptide such as immunogenicity. Preferably, such a sequence is at least 75%, more preferably 80%, and most preferably 90% identical to SEQ ID No: 3 or 4.

[0034] Homologous amino acid sequences include sequences that are identical or substantially identical to SEQ ID No: 3 or 4. By “amino acid sequence substantially identical” is meant a sequence that is at least 90%, preferably 95%, more preferably 97%, and most preferably 99% identical to an amino acid sequence of reference and that preferably differs from the sequence of reference by a majority of conservative amino acid substitutions.

[0035] Conservative amino acid substitutions are substitutions among amino acids of the same class. These classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, such as aspartic acid and glutamic acid; and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.

[0036] Homology is measured using sequence analysis software such as Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705. Amino acid sequences are aligned to maximize identity. Gaps may be artificially introduced into the sequence to attain proper alignment. Once the optimal alignment has been set up, the degree of homology is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions.

[0037] Homologous polynucleotide sequences are defined in a similar way. Preferably, a homologous sequence is one that is at least 45%, more preferably 60%, and most preferably 85% identical to either coding sequence SEQ ID Nos: 1 or 2.

[0038] Consistent with the first aspect of the invention, polypeptides having a sequence homologous to SEQ ID No: 3 or 4 include naturally-occurring allelic variants, as well as mutants or any other non-naturally occurring variants that retain the inherent characteristics of the polypeptide of SEQ ID No: 3 or 4.

[0039] As is known in the art, an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function of the polypeptide. By “biological function” is meant the function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells. For example, the biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium. Biological function is distinct from antigenic property. A polypeptide can have more than one biological function.

[0040] Allelic variants are very common in nature. For example, a bacterial species such as C. pneumoniae, is usually represented by a variety of strains that differ from each other by minor allelic variations. Indeed, a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence (and polynucleotide sequence) that is not identical in each of the strains. Despite this variation, an immune response directed generally against many allelic variants has been demonstrated. In studies of the Chlamydial MOMP antigen, cross-strain antibody binding plus neutralization of infectivity occurs despite amino acid sequence variation of MOMP from strain to strain, indicating that the MOMP, when used as an immunogen, is tolerant of amino acid variations.

[0041] Polynucleotides encoding homologous polypeptides or allelic variants are retrieved by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by conventional methods. This involves the use of synthetic oligonucleotide primers matching upstream and downstream of the 5′ and 3′ ends of the encoding domain. Suitable primers are designed according to the nucleotide sequence information provided in SEQ ID Nos: 1 to 10. The procedure is as follows: a primer is selected which consists of 10 to 40, preferably 15 to 25 nucleotides. It is advantageous to select primers containing C and G nucleotides in a proportion sufficient to ensure efficient hybridization; i.e., an amount of C and G nucleotides of at least 40%, preferably 50% of the total nucleotide content. A standard PCR reaction contains typically 0.5 to 5 Units of Taq DNA polymerase per 100 μL, 20 to 200 μM deoxynucleotide each, preferably at equivalent concentrations, 0.5 to 2.5 MM magnesium over the total deoxynucleotide concentration, 10⁵ to 10⁶ target molecules, and about 20 pmol of each primer. About 25 to 50 PCR cycles are performed, with an annealing temperature 15° C. to 5° C. below the true Tm of the primers. A more stringent annealing temperature improves discrimination against incorrectly annealed primers and reduces incorportion of incorrect nucleotides at the 3′ end of primers. A denaturation temperature of 95° C. to 97° C. is typical, although higher temperatures may be appropriate for dematuration of G+C-rich targets. The number of cycles performed depends on the starting concentration of target molecules, though typically more than 40 cycles is not recommended as non-specific background products tend to accumulate.

[0042] An alternative method for retrieving polynucleotides encoding homologous polypeptides or allelic variants is by hybridization screening of a DNA or RNA library. Hybridization procedures are well-known in the art and are described in Ausubel et al., (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994), Silhavy et al. (Silhavy et al. Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, 1984), and Davis et al. (Davis et al. A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, 1980)). Important parameters for optimizing hybridization conditions are reflected in a formula used to obtain the critical melting temperature above which two complementary DNA strands separate from each other (Casey & Davidson, Nucl. Acid Res. (1977) 4:1539). For polynucleotides of about 600 nucleotides or larger, this formula is as follows: Tm=81.5+0.41×(% G+C)+16.6 log (cation ion concentration)−0.63×(% formamide)−600/base number. Under appropriate stringency conditions, hybridization temperature (Th) is approximately 20 to 40° C., 20 to 25° C., or, preferably 30 to 40° C. below the calculated Tm. Those skilled in the art will understand that optimal temperature and salt conditions can be readily determined.

[0043] For the polynucleotides of the invention, stringent conditions are achieved for both pre-hybridizing and hybridizing incubations (i) within 4-16 hours at 42° C., in 6×SSC containing 50% formamide, or (ii) within 4-16 hours at 65° C. in an aqueous 6×SSC solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)). Typically, hybridization experiments are performed at a temperature from 60 to 68° C., e.g. 65° C. At such a temperature, stringent hybridization conditions can be achieved in 6×SSC, preferably in 2×SSC or 1×SSC, more preferably in 0.5×SSc, 0.3×SSC or 0.1×SSC (in the absence of formamide). 1×SSC contains 0.15 M NaCl and 0.015 M sodium citrate.

[0044] Useful homologs and fragments thereof that do not occur naturally are designed using known methods for identifying regions of an antigen that are likely to tolerate amino acid sequence changes and/or deletions. As an example, homologous polypeptides from different species are compared; conserved sequences are identified. The more divergent sequences are the most likely to tolerate sequence changes. Homology among sequences may be analyzed using the BLAST homology searching algorithm of Altschul et al., Nucleic Acids Res.;25:3389-3402 (1997). Alternatively, sequences are modified such that they become more reactive to T- and/or B-cells, based on computer-assisted analysis of probable T- or B-cell epitopes Yet another alternative is to mutate a particular amino acid residue or sequence within the polypeptide in vitro, then screen the mutant polypeptides for their ability to prevent or treat Chlamydia infection according to the method outlined below.

[0045] A person skilled in the art will readily understand that by following the screening process of this invention, it will be determined without undue experimentation whether a particular homolog of SEQ ID No. 3 or 4 may be useful in the prevention or treatment of Chlamydia infection. The screening procedure comprises the steps:

[0046] (i) immunizing an animal, preferably mouse, with the test homolog or fragment;

[0047] (ii) inoculating the immunized animal with Chlamydia; and

[0048] (iii) selecting those homologs or fragments which confer protection against Chlamydia.

[0049] By “conferring protection” is meant that there is a reduction is severity of any of the effects of Chlamydia infection, in comparison with a control animal which was not immunized with the test homolog or fragment. This section appears as the first part of Example 3 in −3, 2^(nd) priority appl.

[0050] It has been previously demonstrated (Yang, Z. P., Chi, E. Y., Kuo, C. C. and Grayston, J. T. 1993. A mouse model of C. pneumoniae strain TWAR pneumonitis. 61(5):2037-2040) that mice are susceptible to intranasal infection with different isolates of C. pneumoniae. Strain AR-39 (Chi, E. Y., Kuo, C. C. and Grayston, J. T. , 1987. Unique ultrastructure in the elementary body of Chiamydia sp. strain TWAR. J. Bacteriol. 169(8):3757-63) was used in Balb/c mice as a challenge infection model to examine the capacity of chlamydia gene products delivered as naked DNA to elicit a protective response against a sublethal C. pneumoniae lung infection. Protective immunity is defined as an accelerated clearance of pulmonary infection.

[0051] Groups of 7 to 9 week old male Balb/c mice (6 to 10 per group) were immunized intramuscularly (i.m.) plus intranasally (i.n.) with plasmid DNA containing the coding sequence of a C.pneumoniae polypeptide. Saline or the plasmid vector lacking an inserted chlamydial gene was given to groups of control animals.

[0052] For i.m. immunization alternate left and right quadriceps were injected with 100 μg of DNA in 50 μl of PBS on three occasions at 0, 3 and 6 weeks. For i.n. immunization, anaesthetized mice aspirated 50 μl of PBS containing 50 μg DNA on three occasions at 0, 3 and 6 weeks. At week 8, immunized mice were inoculated i.n. with 5×10⁵ IFU of C. pneumoniae , strain AR39 in 100 μl of SPG buffer to test their ability to limit the growth of a sublethal C. pneumoniae challenge.

[0053] Lungs were taken from mice at day 9 post-challenge and immediately homogenised in SPG buffer (7.5% sucrose, 5 mM glutamate, 12.5 mM phosphate pH7.5). The homogenate was stored frozen at −70° C. until assay. Dilutions of the homogenate were assayed for the presence of infectious chlamydia by inoculation onto monolayers of susceptible cells. The inoculum was centrifuged onto the cells at 3000 rpm for 1 hour, then the cells were incubated for three days at 35° C. in the presence of 1 μg/ml cycloheximide. After incubation the monolayers were fixed with formalin and methanol then immunoperoxidase stained for the presence of chlamydial inclusions using convalescent sera from rabbits infected with C.pneumoniae and metal-enhanced DAB as a peroxidase substrate.

[0054] Consistent with the first aspect of the invention, polypeptide derivatives are provided that are partial sequences of SEQ ID No. 3 or 4, partial sequences of polypeptide sequences homologous to SEQ ID No. 3 or 4, polypeptides derived from full-length polypeptides by internal deletion, and fusion proteins.

[0055] It is an accepted practice in the field of immunology to use fragments and variants of protein immunogens as vaccines, as all that is required to induce an immune response to a protein is a small (e.g., 8 to 10 amino acid) immunogenic region of the protein. Various short synthetic peptides corresponding to surface-exposed antigens of pathogens other than Chlamydia have been shown to be effective vaccine antigens against their respective pathogens, e.g. an 11 residue peptide of murine mammary tumor virus (Casey & Davidson, Nucl. Acid Res. (1977) 4:1539), a 16-residue peptide of Semliki Forest virus (Snijders et al., 1991. J. Gen. Virol. 72:557-565), and two overlapping peptides of 15 residues each from canine parvovirus (Langeveld et al., Vaccine 12(15):1473-1480, 1994).

[0056] Accordingly, it will be readily apparent to one skilled in the art, having read the present description, that partial sequences of SEQ ID No: 3 or 4 or their homologous amino acid sequences are inherent to the full-length sequences and are taught by the present invention. Such polypeptide fragments preferably are at least 12 amino acids in length. Advantageously, they are at least 20 amino acids, preferably at least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length.

[0057] Polynucleotides of 30 to 600 nucleotides encoding partial sequences of sequences homologous to SEQ ID No: 3 or 4 are retrieved by PCR amplification using the parameters outlined above and using primers matching the sequences upstream and downstream of the 5′ and 3′ ends of the fragment to be amplified. The template polynucleotide for such amplification is either the full length polynucleotide homologous to one of SEQ ID Nos: 1 to 10, or a polynucleotide contained in a mixture of polynucleotides such as a DNA or RNA library. As an alternative method for retrieving the partial sequences, screening hybridization is carried out under conditions described above and using the formula for calculating Tm. Where fragments of 30 to 600 nucleotides are to be retrieved, the calculated Tm is corrected by subtracting (600/polynucleotide size in base pairs) and the stringency conditions are defined by a hybridization temperature that is 5 to 10° C. below Tm. Where oligonucleotides shorter than 20-30 bases are to be obtained, the formula for calculating the Tm is as follows: Tm=4×(G+C)+2(A+T). For example, an 18 nucleotide fragment of 50% G+C would have an approximate Tm of 54° C. Short peptides that are fragments of SEQ ID No: 3 or 4 or their homologous sequences, are obtained directly by chemical synthesis (E. Gross and H. J. Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; Modern Techniques of Peptide Synthesis, John Wiley & Sons (1981), and M. Bodanzki, Principles of Peptide Synthesis, Springer-Verlag (1984)).

[0058] Useful polypeptide derivatives, e.g., polypeptide fragments, are designed using computer-assisted analysis of amino acid sequences. This would identify probable surface-exposed, antigenic regions (Hughes et al., 1992. Infect. Immun. 60(9):3497). Analysis of 6 amino acid sequences contained in SEQ ID No: 3 or 4, based on the product of flexibility and hydrophobicity propensities using the program SEQSEE (Wishart D S, et al. “SEQSEE: a comprehensive program suite for protein sequence analysis.” Comput Appl Biosci. 1994 April;10(2):121-32), can reveal potential B- and T-cell epitopes which may be used as a basis for selecting useful immunogenic fragments and variants. This analysis uses a reasonable combination of external surface features that is likely to be recognized by antibodies. Probable T-cell epitopes for HLA-A0201 MHC subclass may be revealed by an algorithms that emulate an approach developed at the NIH (Parker K C, et al. “Peptide binding to MHC class I molecules: implications for antigenic peptide prediction.” Immunol Res 1995;14(1):34-57).

[0059] Epitopes which induce a protective T cell-dependent immune response are present throughout the length of the polypeptide. However, some epitopes may be masked by secondary and tertiary structures of the polypeptide. To reveal such masked epitopes large internal deletions are created which remove much of the original protein structure and exposes the masked epitopes. Such internal deletions sometimes effects the additional advantage of removing-immunodominant regions of high variability among strains. Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions are constructed using standard methods (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994). Such methods include standard PCR, inverse PCR, restriction enzyme treatment of cloned DNA molecules, or the method of Kunkel et al. (Kunkel et al. Proc. Natl. Acad. Sci. USA (1985) 82:448). Components for these methods and instructions for their use are readily available from various commercial sources such as Stratagene. Once the deletion mutants have been constructed, they are tested for their ability to prevent or treat Chlamydia infection as described above.

[0060] As used herein, a fusion polypeptide is one that contains a polypeptide or a polypeptide derivative of the invention fused at the N- or C-terminal end to any other polypeptide (hereinafter referred to as a peptide tail). A simple way to obtain such a fusion polypeptide is by translation of an in-frame fusion of the polynucleotide sequences, i.e., a hybrid gene. The hybrid gene encoding the fusion polypeptide is inserted into an expression vector which is used to transform or transfect a host cell. Alternatively, the polynucleotide sequence encoding the polypeptide or polypeptide derivative is inserted into an expression vector in which the polynucleotide encoding the peptide tail is already present. Such vectors and instructions for their use are commercially available, e.g. the pMal-c2 or pMal-p2 system from New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of polypeptides and derivatives of the invention.

[0061] An advantageous example of a fusion polypeptide is one where the polypeptide or homolog or fragment of the invention is fused to a polypeptide having adjuvant activity, such as subunit B of either cholera toxin or E. coli heat-labile toxin. Another advantageous fusion is one where the polypeptide, homolog or fragment is fused to a strong T-cell epitope or B-cell epitope. Such an epitope may be one known in the art (e.g. the Hepatitis B virus core antigen, D. R. Millich et al., “Antibody production to the nucleocapsid and envelope of the Hepatitis B virus primed by a single synthetic T cell site”, Nature. 1987. 329:547-549), or one which has been identified in another polypeptide of the invention based on computer-assisted analysis of probable T- or B-cell epitopes. Consistent with this aspect of the invention is a fusion polypeptide comprising T- or B-cell epitopes from one of SEQ ID No: 3 or 4 or its homolog or fragment, wherein the epitopes are derived from multiple variants of said polypeptide or homolog or fragment, each variant differing from another in the location and sequence of its epitope within the polypeptide. Such a fusion is effective in the prevention and treatment of Chlamydia infection since it optimizes the T- and B-cell response to the overall polypeptide, homolog or fragment.

[0062] To effect fusion, the polypeptide of the invention is fused to the N-, or preferably, to the C-terminal end of the polypeptide having adjuvant activity or T- or B-cell epitope. Alternatively, a polypeptide fragment of the invention is inserted internally within the amino acid sequence of the polypeptide having adjuvant activity. The T- or B-cell epitope may also be inserted internally within the amino acid sequence of the polypeptide of the invention.

[0063] Consistent with the first aspect, the polynucleotides of the invention also encode hybrid precursor polypeptides containing heterologous signal peptides, which mature into polypeptides of the invention. By “heterologous signal peptide” is meant a signal peptide that is not found in naturally-occurring precursors of polypeptides of the invention.

[0064] A polynucleotide molecule according to the invention, including RNA, DNA, or modifications or combinations. thereof, have various applications. A DNA molecule is used, for example, (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating Chlamydia infection, (iii) as a vaccine agent (as well as an RNA molecule), in a naked form or formulated with a delivery vehicle and, (iv) in the construction of attenuated Chlamydia strains that can over-express a polynucleotide of the invention or express it in a non-toxic, mutated form.

[0065] Accordingly, a second aspect of the invention encompasses (i) an expression cassette containing a DNA molecule of the invention placed under the control of the elements required for expression, in particular under the control of an appropriate promoter; (ii) an expression vector containing an expression cassette of the invention; (iii) a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, which involves culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the DNA molecule of the invention and, recovering the encoded polypeptide or polypeptide derivative from the cell culture.

[0066] A recombinant expression system is selected from procaryotic and eucaryotic hosts. Eucaryotic hosts include yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant cells. A preferred expression system is a procaryotic host such as E. coli. Bacterial and eucaryotic cells are available from a number of different sources including commercial sources to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Md.). Commercial sources of cells used for recombinant protein expression also provide instructions for usage of the cells.

[0067] The choice of the expression system depends on the features desired for the expressed polypeptide. For example, it may be useful to produce a polypeptide of the invention in a particular lipidated form or any other form.

[0068] One skilled in the art would redily understand that not all vectors and expression control sequences and hosts would be expected to express equally well the polynucleotides of this invention. With the guidelines described below, however, a selection of vectors, expression control sequences and hosts may be made without undue experimentation and without departing from the scope of this invention.

[0069] In selecting a vector, the host must be chosen that is compatible with the vector which is to exist and possibly replicate in it. Considerations are made with respect to the vector copy number, the ability to control the copy number, expression of other proteins such as antibiotic resistance. In selecting an expression control sequence, a number of variables are considered. Among the important variable are the relative strength of the sequence (e.g. the ability to drive expression under various conditions), the ability to control the sequence's function, compatibility between the polynucleotide to be expressed and the control sequence (e.g. secondary structures are considered to avoid hairpin structures which prevent efficient transcription). In selecting the host, unicellular hosts are selected which are compatible with the selected vector, tolerant of any possible toxic effects of the expressed product, able to secrete the expressed product efficiently if such is desired, to be able to express the product in the desired conformation, to be easily scaled up, and to which ease of purification of the final product.

[0070] The choice of the expression cassette depends on the host system selected as well as the features desired for the expressed polypeptide. Typically, an expression cassette includes a promoter that is functional in the selected host system and can be constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; a region encoding a signal peptide, e.g., a lipidation signal peptide; a DNA molecule of the invention; a stop codon; and optionally a 3′ terminal region (translation and/or transcription terminator). The signal peptide encoding region is adjacent to the polynucleotide of the invention and placed in proper reading frame. The signal peptide-encoding region is homologous or heterologous to the DNA molecule encoding the mature polypeptide and is compatible with the secretion apparatus of the host used for expression. The open reading frame constituted by the DNA molecule of the invention, solely or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system. Promoters and signal peptide encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (as described in U.S. Pat. No. 5,028,530 and in Cagnon et al., (Cagnon et al., Protein Engineering (1991) 4(7):843)); the promoter of the gene of bacteriophage T7 encoding RNA polymerase, that is functional in a number of E. coli strains expressing T7 polymerase (described in U.S. Pat. No. 4,952,496); OspA lipidation signal peptide ; and RlpB lipidation signal peptide (Takase et al., J. Bact. (1987) 169:5692).

[0071] The expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system. Expression vectors (e.g., plasmids or viral vectors) can be chosen, for example, from those described in Pouwels et al. (Cloning Vectors: A Laboratory Manual 1985, Supp. 1987). Suitable expression vectors can be purchased from various commercial sources.

[0072] Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected as described in Ausubel et al., (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994).

[0073] Upon expression, a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane. The polypeptide is recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the recombinant cell culture. Typically, the recombinant polypeptide is purified by antibody-based affinity purification or by other well-known methods that can be readily adapted by a person skilled in the art, such as fusion of the polynucleotide encoding the polypeptide or its derivative to a small affinity binding domain. Antibodies useful for purifying by immunoaffinity the polypeptides of the invention are obtained as described below.

[0074] A polynucleotide of the invention can also be useful as a vaccine. There are two major routes, either using a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention is evaluated as described below.

[0075] Accordingly, a third aspect of the invention provides (i) a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression; (ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; specifically (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against Chlamydia in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing Chlamydia infection of animals, e.g., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit a protective or therapeutic immune response to Chlamydia; and particularly, (v) a method for preventing and/or treating a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumonia, C. pecorum) infection, which involves administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an infected individual. Additionally, the third aspect of the invention encompasses the use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.

[0076] As used herein, a vaccine vector expresses one or several polypeptides or derivatives of the invention, The vaccine vector may express additionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the immune response (adjuvant effect). It is understood that each of the components to be expressed is placed under the control of elements required for expression in a mammalian cell.

[0077] Consistent with the third aspect of the invention is a composition comprising several vaccine vectors, each of them capable of expressing a polypeptide or derivative of the invention. A composition may also comprise a vaccine vector capable of expressing an additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof; optionally together with or a cytokine such as IL-2 or IL-12.

[0078] Vaccination methods for treating or preventing infection in a mammal comprises use of a vaccine vector of the invention to be administered by any conventional route particularly to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. Preferred routes depend upon the choice of the vaccine vector. Treatment may be effected in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by skilled artisans such as the vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated (weight, age and the like).

[0079] Live vaccine vectors available in the art include viral vectors such as adenoviruses and poxviruses as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilié de Calmette-Guérin (BCG), and Streptococcus.

[0080] An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a DNA molecule of the invention, are described in U.S. Pat. No. 4,920,209. Poxvirus vectors include vaccinia and canary pox virus, described in U.S. Pat. No. 4,722,848 and U.S. Pat. No. 5,364,773, respectively. (Also see, e.g., Tartaglia et al., Virology (1992) 188:217) for a description of a vaccinia virus vector and-Taylor et al, Vaccine (1995) 13:539 for a reference of a canary pox.) Poxvirus vectors capable of expressing a polynucleotide of the invention are obtained by homologous recombination as described in Kieny et al., Nature (1984) 312:163 so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells. Generally, the dose of vaccine viral vector, for therapeutic or prophylactic use, can be of from about 1×10⁴ to about 1×10¹¹, advantageously from about 1×10⁷ to about 1×10¹, preferably of from about 1×10⁷ to about 1×10⁹ plaque-forming units per kilogram. Preferably, viral vectors are administered parenterally; for example, in 3 doses, 4 weeks apart. It is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself.

[0081] Non-toxicogenic Vibrio cholerae mutant strains that are useful as a live oral vaccine are known. Mekalanos et al., Nature (1983) 306:551 and U.S. Pat. No. 4,882,278 describe strains which have a substantial amount of the coding sequence of each of the two ctxA alleles deleted so that no functional cholerae toxin is produced. WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations. WO 94/01533 describes a deletion mutant lacking functional ctxA and attRS1 DNA sequences. These mutant strains are genetically engineered to express heterologous antigens, as described in WO 94/19482. An effective vaccine dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention contains about 1×10⁵ to about 1×10⁹, preferably about 1×10⁶ to about 1×10⁸, viable bacteria in a volume appropriate for the selected route of administration. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.

[0082] Attenuated Salmonella typhimurium strains, genetically engineered for recombinant expression of heterologous antigens or not, and their use as oral vaccines are described in Nakayama et al. (Bio/Technology (1988) 6:693) and WO 92/11361. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.

[0083] Other bacterial strains used as vaccine vectors in the context of the present invention are described for Shigella flexneri in High et al., EMBO (1992) 11:1991 and Sizemore et al., Science (1995) 270:299; for Streptococcus gordonii in Medaglini et al., Proc. Natl. Acad. Sci. USA (1995) 92:6868; and for Bacille Calmette Guerin in Flynn J. L., Cell. Mol. Biol. (1994) 40 (suppl. I):31, WO 88/06626, WO 90/00594, WO 91/13157, WO 92/01796, and WO 92/21376.

[0084] In bacterial vectors, the polynucleotide of the invention is inserted into the bacterial genome or remains in a free state as part of a plasmid.

[0085] The composition comprising a vaccine bacterial vector of the present invention may further contain an adjuvant. A number of adjuvants are known to those skilled in the art. Preferred adjuvants are selected as provided below.

[0086] Accordingly, a fourth aspect of the invention provides (i) a composition of matter comprising a polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a polynucleotide of the invention; (iii) a method for inducing an immune response against Chlamydia in a mammal by administration of an immunogenically effective amount of a polynucleotide of the invention to elicit a protective immune response to Chlamydia; and particularly, (iv) a method for preventing and/or treating a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae, or C. pecorum) infection, by administering a prophylactic or therapeutic amount of a polynucleotide of the invention to an infected individual. Additionally, the fourth aspect of the invention encompasses the use of a polynucleotide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection. A preferred use includes the use of a DNA molecule placed under conditions for expression in a mammalian cell, especially in a plasmid that is unable to replicate in mammalian cells and to substantially integrate in a mammalian genome.

[0087] Use of the polynucleotides of the invention include their administration to a mammal as a vaccine, for therapeutic or prophylactic purposes. Such polynucleotides are used in the form of DNA as part of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome. Typically, such a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell. The promoter functions either ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S. Pat. No. 4,168,062) and the Rous Sarcoma Virus promoter (described in Norton & Coffin, Molec. Cell Biol. (1985) 5:281). An example of a tissue-specific promoter is the desmin promoter which drives expression in muscle cells (Li et al., Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991) 266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403). Use of promoters is well-known to those skilled in the art. Useful vectors are described in numerous publications, specifically WO 94/21797 and Hartikka et al., Human Gene Therapy (1996) 7:1205.

[0088] Polynucleotides of the invention which are used as a vaccine encode either a precursor or a mature form of the corresponding polypeptide. In the precursor form, the signal peptide is either homologous or heterologous. In the latter case, a eucaryotic leader sequence such as the leader sequence of the tissue-type plasminogen factor (tPA) is preferred.

[0089] As used herein, a composition of the invention contains one or several polynucleotides with optionally at least one additional polynucleotide encoding another Chlamydia antigen such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof. The composition may also contain an additional polynucleotide encoding a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) so that the immune response is enhanced. These additional polynucleotides are placed under appropriate control for expression. Advantageously, DNA molecules of the invention and/or additional DNA molecules to be included in the same composition, are present in the same plasmid.

[0090] Standard techniques of molecular biology for preparing and purifying polynucleotides are used in the preparation of polynucleotide therapeutics of the invention. For use as a vaccine, a polynucleotide of the invention is formulated according to various methods outlined below.

[0091] One method utililizes the polynucleotide in a naked form, free of any delivery vehicles. Such a polynucleotide is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, with or without a carrier. When present, the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose.

[0092] An alternative method utilizes the polynucleotide in association with agents that assist in cellular uptake. Examples of such agents are (i) chemicals that modify cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) liposomes for encapsulation of the polynucleotide, or (iii) cationic lipids or silica, gold, or tungsten microparticles which associate themselves with the polynucleotides.

[0093] Anionic and neutral liposomes are well-known in the art (see, e.g., Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides. Cationic lipids are also known in the art and are commonly used for gene delivery. Such lipids include Lipofectin™ also known as DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol). A description of these cationic lipids can be found in EP 187,702, WO 90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. Pat. No. 5,527,928. Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine), as described in WO 90/11092 as an example.

[0094] Formulation containing cationic liposomes may optionally contain other transfection-facilitating compounds. A number of them are described in WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/02397. They include spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).

[0095] Gold or tungsten microparticles are used for gene delivery, as described in WO 91/00359, WO 93/17706, and Tang et al. Nature (1992) 356:152. The microparticle-coated polynucleotide is injected via intradermal or intraepidermal routes using a needleless injection device (“gene gun”), such as those described in U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,015,580, and WO 94/24263.

[0096] The amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal), the mode of administration, and the type of formulation. In general, a therapeutically or prophylactically effective dose from about 1 μg to about 1 mg, preferably, from about 10 μg to about 800 μg and, more preferably, from about 25 μg to about 250 μg, can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals.

[0097] The route of administration is any conventional route used in the vaccine field. As general guidance, a polynucleotide of the invention is administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route. The choice of administration route depends on the formulation that is selected. A polynucleotide formulated in association with bupivacaine is advantageously administered into muscles. When a neutral or anionic liposome or a cationic lipid, such as DOTMA or DC-Chol, is used, the formulation can be advantageously injected via intravenous, intranasal (aerosolization), intramuscular, intradermal, and subcutaneous routes. A polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or sub-cutaneous routes.

[0098] Although not absolutely required, such a composition can also contain an adjuvant. If so, a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e.g., QS21, which is described in U.S. Pat. No. 5,057,546.

[0099] The sequence information provided in the present application enables the design of specific nucleotide probes and primers that are used for diagnostic purposes. Accordingly, a fifth aspect of the invention provides a nucleotide probe or primer having a sequence found in or derived by degeneracy of the genetic code from a sequence shown in SEQ ID No: 1 or 2.

[0100] The term “probe” as used in the present application refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to nucleic acid molecules having SEQ ID No: 1 or 2 or to sequences homologous to SEQ ID No: 1 or 2, or to their complementary or anti-sense sequences. Generally, probes are significantly shorter than full-length sequences. Such probes contain from about 5 to about 100, preferably from about 10 to about 80, nucleotides. In particular, probes have sequences that are at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of SEQ ID No: 1 or 2 or that are complementary to such sequences. Probes may contain modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2,6-purine. Sugar or phosphate residues may also be modified or substituted. For example, a deoxyribose residue may be replaced by a polyamide (Nielsen et al., Science (1991) 254:1497) and phosphate residues may be replaced by ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters. In addition, the 2′-hydroxyl group on ribonucleotides may be modified by including such groups as alkyl groups.

[0101] Probes of the invention are used in diagnostic tests, as capture or detection probes. Such capture probes are conventionally immobilized on a solid support, directly or indirectly, by covalent means or by passive adsorption. A detection probe is labelled by a detection marker selected from: radioactive isotopes, enzymes such as peroxidase, alkaline phosphatase, and enzymes able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate, compounds that are chromogenic, fluorogenic, or luminescent, nucleotide base analogs, and biotin.

[0102] Probes of the invention are used in any conventional hybridization technique, such as dot blot (Maniatis et al., Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), Southern blot (Southern, J. Mol. Biol. (1975) 98:503), northern blot (identical to Southern blot with the exception that RNA is used as a target), or the sandwich technique (Dunn et al., Cell (1977) 12:23). The latter technique involves the use of a specific capture probe and/or a specific detection probe with nucleotide sequences that at least partially differ from each other.

[0103] A primer is a probe of usually about 10 to about 40 nucleotides that is used to initiate enzymatic polymerization of DNA in an amplification process (e.g., PCR), in an elongation process, or in a reverse transcription method. Primers used in diagnostic methods involving PCR are labeled by methods known in the art.

[0104] As described herein, the invention also encompasses (i) a reagent comprising a probe of the invention for detecting and/or identifying the presence of Chlamydia in a biological material; (ii) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA or RNA is extracted from the material and denatured, and (c) exposed to a probe of the invention, for example, a capture, detection probe or both, under stringent hybridization conditions, such that hybridization is detected; and (iii) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is primed with at least one, and preferably two, primers of the invention and amplified by polymerase chain reaction, and (d) the amplified DNA fragment is produced.

[0105] It is apparent that disclosure of polynucleotide sequences of SEQ ID No: 1 or 2, their homolog, and partial sequences of either enable their corresponding amino acid sequences. Accordingly, a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention.

[0106] A “substantially purified polypeptide” as used herein is defined as a polypeptide that is separated from the environment in which it naturally occurs and/or that is free of the majority of the polypeptides that are present in the environment in which it was synthesized. For example, a substantially purified polypeptide is free from cytoplasmic polypeptides. Those s-killed in the art would readily understand that the polypeptides of the invention may be purified from a natural source, i.e., a Chlamydia strain, or produced by recombinant means.

[0107] Consistent with the sixth aspect of the invention are polypeptides, homologs or fragments which are modified or treated to enhance their immunogenicity in the target animal, in whom the polypeptide, homolog or fragments are intended to confer protection against Chlamydia. Such modifications or treatments include: amino acid substitutions with an amino acid derivative such as 3-methyhistidine, 4-hydroxyproline, 5-hydroxylysine etc., modifications or deletions which are carried out after preparation of the polypeptide, homolog or fragment, such as the modification of free amino, carboxyl or hydroxyl side groups of the amino acids.

[0108] Identification of homologous polypeptides or polypeptide derivatives encoded by polynucleotides of the invention which have specific antigenicity is achieved by screening for cross-reactivity with an antiserum raised against the polypeptide of reference having an amino acid sequence of any one of SEQ ID No: 3 or 4. The procedure is as follows: a monospecific hyperimmune antiserum is raised against a purified reference polypeptide, a fusion polypeptide (for example, an expression product of MBP, GST, or His-tag systems, the description and instructions for use of which are contained in Invitrogen product manuals for pcDNA3.1/Myc-His(+) A, B, and C and for the Xpress™ System Protein Purification), or a synthetic peptide predicted to be antigenic. Where an antiserum is raised against a fusion polypeptide, two different fusion systems are employed. Specific antigenicity can be determined according to a number of methods, including Western blot (Towbin et al., Proc. Natl. Acad. Sci. USA (1979) 76:4350), dot blot, and ELISA, as described below.

[0109] In a Western blot assay, the product to be screened, either as a purified preparation or a total E. coli extract, is submitted to SDS-Page electrophoresis as described by Laemmli (Nature (1970) 227:680). After transfer to a nitrocellulose membrane, the material is further incubated with the monospecific hyperimmune antiserum diluted in the range of dilutions from about 1:5 to about 1:5000, preferably from about 1:100 to about 1:500. Specific antigenicity is shown once a band corresponding to the product exhibits reactivity at any of the dilutions in the above range.

[0110] In an ELISA assay, the product to be screened is preferably used as the coating antigen. A purified preparation is preferred, although a whole cell extract can also be used. Briefly, about 100 μl of a preparation at about 10 μg protein/ml are distributed into wells of a 96-well polycarbonate ELISA plate. The plate is incubated for 2 hours at 37° C. then overnight at 4° C. The plate is washed with phosphate buffer saline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer). The wells are saturated with 250 μl PBS containing 1% bovine serum albumin (BSA) to prevent non-specific antibody binding. After 1 hour incubation at 37° C., the plate is washed with PBS/Tween buffer. The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA. 100 μl of dilutions are added per well. The plate is incubated for 90 minutes at 37° C., washed and evaluated according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when specific antibodies were raised in rabbits. Incubation is carried out for 90 minutes at 37° C. and the plate is washed. The reaction is developed with the appropriate substrate and the reaction is measured by colorimetry (absorbance measured spectrophotometrically). Under the above experimental conditions, a positive reaction is shown by O.D. values greater than a non immune control serum.

[0111] In a dot blot assay, a purified product is preferred, although a whole cell extract can also be used. Briefly, a solution of the product at about 100 μg/ml is serially two-fold diluted in 50 mM Tris-HCl (pH 7.5). 100 μl of each dilution are applied to a nitrocellulose membrane 0.45 μm set in a 96-well dot blot apparatus (Biorad). The buffer is removed by applying vacuum to the system. Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried. The membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M NaCl, 10 g/L skim milk) and incubated with an antiserum dilution from about 1:50 to about 1:5000, preferably about 1:500. The reaction is revealed according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when rabbit antibodies are used. Incubation is carried out 90 minutes at 37° C. and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is measured visually by the appearance of a colored spot! e.g., by colorimetry. Under the above experimental conditions, a positive reaction is shown once a colored spot is associated with a dilution of at least about 1:5, preferably of at least about 1:500.

[0112] Therapeutic or prophylactic efficacy of a polypeptide or derivative of the invention can be evaluated as described below. A seventh aspect of the invention provides (i) a composition of matter comprising a polypeptide of the invention together with a diluent or carrier; specifically (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention; (iii) a method for inducing an immune response against Chlamydia in a mammal, by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit a protective immune response to Chlamydia; and particularly, (iv) a method for preventing and/or treating a Chlamydia (e.g., C. trachomatis. C. psittaci, C. pneumoniae. or C. pecorum) infection, by administering a prophylactic or therapeutic amount of a polypeptide of the invention to an infected individual. Additionally, the seventh aspect of the invention encompasses the use of a polypeptide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.

[0113] As used herein, the immunogenic compositions of the invention are administered by conventional routes known the vaccine field, in particular to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. The choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, the intranasal or oral route is preferred. If a lipid formulation or an aluminum compound is used, the parenteral route is preferred with the sub-cutaneous or intramuscular route being most preferred. The choice also depends upon the nature of the vaccine agent. For example, a polypeptide of the invention fused to CTB or LTB is best administered to a mucosal surface.

[0114] As used herein, the composition of the invention contains one or several polypeptides or derivatives of the invention. The composition optionally contains at least one additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof.

[0115] For use in a composition of the invention, a polypeptide or derivative thereof is formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response. These compounds are readily available to one skilled in the art; for example, see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990).

[0116] Adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants may protect the antigen from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. An appropriate selection can conventionally be made by those skilled in the art, for example, from those described below (under the eleventh aspect of the invention).

[0117] Treatment is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art. For example, a priming dose is followed by three booster doses at weekly or monthly intervals. An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art. In general, a vaccine antigen of the invention is administered by a mucosal route in an amount from about 10 μg to about 500 mg, preferably from about 1 mg to about 200 mg. For the parenteral route of administration, the dose usually does not exceed about 1 mg, preferably about 100 μg.

[0118] When used as vaccine agents, polynucleotides and polypeptides of the invention may be used sequentially as part of a multistep immunization process. For example, a mammal is initially primed with a vaccine vector of the invention such as a pox virus, e.g., via the parenteral route, and then boosted twice with the polypeptide encoded by the vaccine vector, e.g., via the mucosal route. In another example, liposomes associated with a polypeptide or derivative of the invention is also used for priming, with boosting being carried out mucosally using a soluble polypeptide or derivative of the invention in combination with a mucosal adjuvant (e.g., LT).

[0119] A polypeptide derivative of the invention is also used in accordance with the seventh aspect as a diagnostic reagent for detecting the presence of anti-Chlamydia antibodies, e.g., in a blood sample. Such polypeptides are about 5 to about 80, preferably about 10 to about 50 amino acids in length. They are either labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.

[0120] Upon expression of a DNA molecule of the invention, a polypeptide or polypeptide derivative is produced and purified using known laboratory techniques. As described above, the polypeptide or polypeptide derivative may be produced as a fusion protein containing a fused tail that facilitates purification. The fusion product is used to immunize a small mammal, e.g., a mouse or a rabbit, in order to raise antibodies against the polypeptide or polypeptide derivative (monospecific antibodies). Accordingly, an eighth aspect of the invention provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.

[0121] By “monospecific antibody” is meant an antibody that is capable of reacting with a unique naturally-occurring Chlamydia polypeptide. An antibody of the invention is either polyclonal or monoclonal. Monospecific antibodies may be recombinant, e.g., chimeric (e.g., constituted by a variable region of murine origin associated with a human constant region), humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e.g., murine, origin), and/or single chain. Both polyclonal and monospecific antibodies may also be in the form of immunoglobulin fragments, e.g., F(ab)′2 or Fab fragments. The antibodies of the invention are of any isotype, e.g., IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.

[0122] Antibodies against the polypeptides, homologs or fragments of the present invention are generated by immunization of a mammal with a composition comprising said polypeptide, homolog or fragment. Scu antibodies may be polyclonal or monoclonal. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see “Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Eds. E. Harlow and D. Lane (1988), and D. E. Yelton et al., 1981. Ann. Rev. Biochem. 50:657-680. For monoclonal antibodies, see Kohler & Milstein (1975) Nature 256:495-497.

[0123] The antibodies of the invention, which are raised to a polypeptide or polypeptide derivative of the invention, are produced and identified using standard immunological assays, e.g., Western blot analysis, dot blot assay, or ELISA (see, e.g., Coligan et al., Current Protocols in Immunology (1994) John Wiley & Sons, Inc., New York, N.Y.). The antibodies are used in diagnostic methods to detect the presence of a Chlamydia antigen in a sample, such as a biological sample. The antibodies are also used in affinity chromatography for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, such antibodies may be used in prophylactic and therapeutic passive immunization methods.

[0124] Accordingly, a ninth aspect of the invention provides (i) a reagent for detecting the presence of Chlamydia in a biological sample that contains an antibody, polypeptide, or polypeptide derivative of the invention; and (ii) a diagnostic method for detecting the presence of Chlamydia in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative of the invention, such that an immune complex is formed, and by detecting such complex to indicate the presence of Chlamydia in the sample or the organism from which the sample is derived.

[0125] Those skilled in the art will readily understand that the immune complex is formed between a component of the sample and the antibody, polypeptide, or polypeptide derivative, whichever is used, and that any unbound material is removed prior to detecting the complex. It is understood that a polypeptide reagent is useful for detecting the presence of anti-Chlamydia antibodies in a sample, e.g., a blood sample, while an antibody of the invention is used for screening a sample, such as a gastric extract or biopsy, for the presence of Chlamydia polypeptides.

[0126] For diagnostic applications, the reagent (i.e., the antibody, polypeptide, or polypeptide derivative of the invention) is either in a free state or immobilized on a solid support, such as a tube, a bead, or any other conventional support used in the field. Immobilization is achieved using direct or indirect means. Direct means include passive adsorption (non-covalent binding) or covalent binding between the support and the reagent. By “indirect means” is meant that an anti-reagent compound that interacts with a reagent is first attached to the solid support. For example, if a polypeptide reagent is used, an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not involved in the recognition of antibodies in biological samples. Indirect means may also employ a ligand-receptor system, for example, where a molecule such as a vitamin is grafted onto the polypeptide reagent and the corresponding receptor immobilized on the solid phase. This is illustrated by the biotin-streptavidin system. Alternatively, a peptide tail is added chemically or by genetic engineering to the reagent and the grafted or fused product immobilized by passive adsorption or covalent linkage of the peptide tail.

[0127] Such diagnostic agents may be included in a kit which also comprises instructions for use. The reagent are labeled with a detection means which allows for the detection of the reagent when it is bound to its target. The detection means may be a fluorescent agent such as fluorescein isocyanate or fluorescein isothiocyanate, or an enzyme such as horse radish peroxidase or luciferase or alkaline phosphatase, or a radioactive element such as ¹²⁵I or ⁵¹Cr.

[0128] Accordingly, a tenth aspect of the invention provides a process for purifying, from a biological sample, a polypeptide or polypeptide derivative of the invention, which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody of the invention.

[0129] For use in a purification process of the invention, the antibody is either polyclonal or monospecific, and preferably is of the IgG type. Purified IgGs is prepared from an antiserum using standard methods (see, e.g., Coligan et al., Current Protocols in Immunology (1994)John Wiley & Sons, Inc., New York, N.Y.). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described in, e.g., Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds. (1988) and outlined below.

[0130] Briefly, a biological sample, such as an C. pneumoniae extract preferably in a buffer solution, is applied to a chromatography material, preferably equilibrated with the buffer used to dilute the biological sample so that the polypeptide or polypeptide derivative of the invention (i.e., the antigen) is allowed to adsorb onto the material. The chromatography material, such as a gel or a resin coupled to an antibody of the invention, is in either a batch form or a column. The unbound components are washed off and the antigen is then eluted with an appropriate elution buffer, such as a glycine buffer or a buffer containing a chaotropic agent, e.g., guanidine HCl, or high salt concentration (e.g., 3 M MgCl₂). Eluted fractions are recovered and the presence of the antigen is detected, e.g., by measuring the absorbance at 280 nm.

[0131] An eleventh aspect of the invention provides (i) a composition of matter comprising a monospecific antibody of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a monospecific antibody of the invention, and (iii) a method for treating or preventing a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae or C. pecorum) infection, by administering a therapeutic or prophylactic amount of a monospecific antibody of the invention to an infected individual. Additionally, the eleventh aspect of the invention encompasses the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing Chlamydia infection.

[0132] The monospecific antibody is either polyclonal or monoclonal, preferably of the IgA isotype (predominantly). In passive immunization, the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, e.g., orally or intragastrically, advantageously, in the presence of a bicarbonate buffer. Alternatively, systemic administration, not requiring a bicarbonate buffer, is carried out. A monospecific antibody of the invention is administered as a single active component or as a mixture with at least one monospecific antibody specific for a different Chlamydia polypeptide. The amount of antibody and the particular regimen used are readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibodies over one week, or three doses per day of about 100 to 1,000 mg of antibodies over two or three days, are effective regimens for most purposes.

[0133] Therapeutic or prophylactic efficacy are evaluated using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the C. pneumoniae mouse model. Those skilled in the art will readily recognize that the C. pneumoniae strain of the model may be replaced with another Chlamydia strain. For example, the efficacy of DNA molecules and polypeptides from C. pneumoniae is preferably evaluated in a mouse model using C. pneumoniae strain. Protection is determined by comparing the degree of Chlamydia infection to that of a control group. Protection is shown when infection is reduced by comparison to the control group. Such an evaluation is made for polynucleotides, vaccine vectors, polypeptides and derivatives thereof, as well as antibodies of the invention.

[0134] Adjuvants useful in any of the vaccine compositions described above are as follows.

[0135] Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, Mont.), is used in parenteral administration.

[0136] Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB). Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/06627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants, such as a bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, or polylactide glycolide (PLGA) microspheres, is also be used in mucosal administration.

[0137] Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415), DC-chol (3 b-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol; U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (WO 88/09336).

[0138] Any pharmaceutical composition of the invention containing a polynucleotide, a polypeptide, a polypeptide derivative, or an antibody of the invention, is manufactured in a conventional manner. In particular, it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF.

[0139] The invention also includes methods in which Chlamiydia infection are treated by oral administration of a Chlamydia polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antacid, sucralfate, or a combination thereof. Examples of such compounds that can be administered with the vaccine antigen and the adjuvant are antibiotics, including, e.g., macrolides, tetracyclines, and derivatives thereof (specific examples of antibiotics that can be used include azithromycin or doxicyclin or immunomodulators such as cytokines or steroids). In addition, compounds containing more than one of the above-listed components coupled together, are used. The invention also includes compositions for carrying out these methods, i.e., compositions containing a Chlamydia antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent.

[0140] It has recently been shown that the 60kDa cysteine rich membrane protein contains a sequence cross-reactive with the murine alpha-myosin heavy chain epitope M7A-alpha, an epitope conserved in humans (Bachmaier et al., Science (1999) 283:1335). This cross-reactivity is proposed to contribute to the development of cardiovascular disease, so it may be beneficial to remove this epitope, and any other epitopes cross-reactive with human antigens, from the protein if it is to be used as a vaccine. Accordingly, a further embodiment of the present invention includes the modification of the coding sequence, for example, by deletion or substitution of the nucleotides encoding the epitope from polynucleotides encoding the protein, as to improve the efficacy and safety of the protein as a vaccine. A similar approach may be appropriate for any protective antigen found to have unwanted homologies or cross-reactivities with human antigens.

[0141] Amounts of the above-listed compounds used in the methods and compositions of the invention are readily determined by one skilled in the art. Treatment/immunization schedules are also known and readily designed by one skilled in the art. For example, the non-vaccine components can be administered on days 1-14, and the vaccine antigen+adjuvant can be administered on days 7, 14, 21, and 28.

EXAMPLES

[0142] The above disclosure generally discribes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Example 1

[0143] This example illustrates the preparation of the eukaryotic expression vector pCA/Myc-His.

[0144] Plasmid pcDNA3.1(−)Myc-His C (Invitrogen) was restricted with Spe I and Bam HI to remove the CMV promoter and the remaining vector fragment was isolated. The CMV promoter and intron A from plasmid VR-1012 (Vical) was isolated on a Spe I/Bam HI fragment. The fragments were ligated together to produce plasmid pCA/Myc-His.

Example 2

[0145] This example illustrates the preparation of a plasmid expression vector containing the 98 kDa outer membrane protein gene.

[0146] The 98 kDa outer membrane protein gene was amplified from Chlamydia pneumoniae genomic DNA by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGGCAGAGGTGACCTTAGATAG 3′) (SEQ ID No: 4) and a 3′ primer (5° CGGCTCGAGTGAAACAAAACTTAGAGCCTAG 3′) (SEQ ID No: 5). The 5′ primer contains a Not I restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the 98kDa outer membrane protein coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the 98 kDa outer membrane protein gene and a Xho I restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.

[0147] After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen) and then digested with Not I and Xho I. The Not I/Xho I restricted PCR fragment containing the 98 kDa outer membrane protein gene was ligated into the Not I and Xho I restricted plasmid pCA/Myc-His to produce plasmid pCAI640 (FIG. 3). Transcription of the 98KDa outer membrane protein gene in pCAI640 was under control of the human cytomegalovirus (CMV) promoter.

[0148] The plasmid pCAI640, was transferred by electroporation into E. coli XL-1 blue (Stratagene) which was grown in LB broth containing 50 μg/ml of carbenicillin. The plasmid was isolated by Endo Free Plasmid Giga Kit™ (Qiagen) large scale DNA purification system. DNA concentration was determined by absorbance at 260 nm and the plasmid was verified after gel electrophoresis and ethidium bromide staining and comparison to molecular weight standards. The 5′ and 3′ ends of the gene were 30 verified by sequencing using a LiCor model 4000 L DNA sequencer and IRD-800 labelled primers.

Example 3

[0149] This example illustrates the immunization of mice to achieve protection against an intranasal challenge of C. pneumoniae.

[0150] It has been previously demonstrated (Yang, Z. P., Chi, E. Y., Kuo, C. C. and Grayston, J. T. 1993. A mouse model of C. pneumoniae strain TWAR pneumonitis. 61(5):2037-2040) that mice are susceptible to intranasal infection with different isolates of C. pneumoniae. Strain AR-39 (Chi, E. Y., Kuo, C. C. and Grayston, J. T. , 1987. Unique ultrastructure in the elementary body of Chlamydia sp. strain TWAR. J. Bacteriol. 169(8):3757-63) was used in Balb/c mice as a challenge infection model to examine the capacity of chlamydia gene products delivered as naked DNA to elicit a protective response against a sublethal C. pneumoniae lung infection. Protective immunity is defined as an accelerated clearance of pulmonary infection.

[0151] Groups of 7 to 9 week old male Balb/c mice (8 to 10 per group) were immunized intramuscularly (i.m.) plus intranasally (i.n.) with plasmid DNA containing the coding sequence of C.pneumoniae 98 kDa outer membrane protein gene as described in Example 1 and 2. Saline or the plasmid vector lacking an inserted chlamydial gene was given to groups of control animals.

[0152] For i.m. immunization alternate left and right quadriceps were injected with 100 μg of DNA in 50 μl of PBS on three occasions at 0, 3 and 6 weeks. For i.n. immunization, anaesthetized mice aspirated 50 μl of PBS containing 50 μg DNA on three occasions at 0, 3 and 6 weeks. At week 8, immunized mice were inoculated i.n. with 5×10⁵ IFU of C. pneumoniae , strain AR39 in 100 μl of SPG buffer to test their ability to limit the growth of a sublethal C. pneumoniae challenge.

[0153] Lungs were taken from mice at days 5 and 9 post-challenge and immediately homogenised in SPG buffer (7.5% sucrose, 5 mM glutamate, 12.5mM phosphate pH7.5). The homogenate was stored frozen at −70° C. until assay. Dilutions of the homogenate were assayed for the presence of infectious chlamydia by inoculation onto monolayers of susceptible cells. The inoculum was centrifuged onto the cells at 3000 rpm for 1 hour, then the cells were incubated for three days at 35° C. in the presence of 1 μg/ml cycloheximide. After incubation the monolayers were fixed with formalin and methanol then immunoperoxidase stained for the presence of chlamydial inclusions using convalescent sera from rabbits infected with C.pneumoniae and metal-enhanced DAB as a peroxidase substrate.

[0154]FIG. 4 shows that mice immunized i.n. and i.m. with pCAI640 had chlamydial lung titers less than 255,000 in 4 of 4 cases at day 5 and less than 423,200 in 4 of 4 cases at day 9 whereas the range of values for control mice sham immunized with saline was 227,000-934,200 IFU/lung (mean 685,240) at day 5 and 96,000-494,000 IFU/lung (mean 238,080) at day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCAI634, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice. The construct pCAI634 is identical to pCAI640 except that the nucleotide sequence encoding the 98 kDa outer membrane protein gene is replaced with a C. pneumoniae nucleotide sequence encoding a different putative 98 kDa outer membrane protein.

1 6 1 3050 DNA Chlamydia pneumoniae CDS (101)..(2908) 1 gattctccgc atcaatcaat tccttgcgtt tcccttgatt tctttttttc tttacagtat 60 ttgctaattt aatttccttg tttcaaaaaa gtgcttacaa atg aag tcc tct gtc 115 Met Lys Ser Ser Val 1 5 tct tgg ttg ttc ttt tct tca atc ccg ctc ttt tca tcg ctc tct ata 163 Ser Trp Leu Phe Phe Ser Ser Ile Pro Leu Phe Ser Ser Leu Ser Ile 10 15 20 gtc gcg gca gag gtg acc tta gat agc agc aat aat agc tat gat gga 211 Val Ala Ala Glu Val Thr Leu Asp Ser Ser Asn Asn Ser Tyr Asp Gly 25 30 35 tct aac gga act acc ttc acg gtc ttt tcc act acg gac gct gct gca 259 Ser Asn Gly Thr Thr Phe Thr Val Phe Ser Thr Thr Asp Ala Ala Ala 40 45 50 gga act acc tat tcc tta ctt tcc gac gta tcc ttt caa aat gca ggg 307 Gly Thr Thr Tyr Ser Leu Leu Ser Asp Val Ser Phe Gln Asn Ala Gly 55 60 65 gct tta gga att ccc tta gcc tca gga tgc ttc cta gaa gcg ggc ggc 355 Ala Leu Gly Ile Pro Leu Ala Ser Gly Cys Phe Leu Glu Ala Gly Gly 70 75 80 85 gat ctt act ttc caa gga aat caa cat gca ctg aag ttt gca ttt atc 403 Asp Leu Thr Phe Gln Gly Asn Gln His Ala Leu Lys Phe Ala Phe Ile 90 95 100 aat gcg ggc tct agc gct gga act gta gcc agt acc tca gca gca gat 451 Asn Ala Gly Ser Ser Ala Gly Thr Val Ala Ser Thr Ser Ala Ala Asp 105 110 115 aag aat ctt ctc ttt aat gat ttt tct aga ctc tct att atc tct tgt 499 Lys Asn Leu Leu Phe Asn Asp Phe Ser Arg Leu Ser Ile Ile Ser Cys 120 125 130 ccc tct ctt ctt ctc tct cct act gga caa tgt gct tta aaa tct gtg 547 Pro Ser Leu Leu Leu Ser Pro Thr Gly Gln Cys Ala Leu Lys Ser Val 135 140 145 ggg aat cta tct cta act ggc aat tcc caa att ata ttt act cag aac 595 Gly Asn Leu Ser Leu Thr Gly Asn Ser Gln Ile Ile Phe Thr Gln Asn 150 155 160 165 ttc tcg tca gat aac ggc ggt gtt atc aat acg aaa aac ttc tta tta 643 Phe Ser Ser Asp Asn Gly Gly Val Ile Asn Thr Lys Asn Phe Leu Leu 170 175 180 tca ggg aca tct cag ttt gcg agc ttt tcg aga aac caa gcc ttc aca 691 Ser Gly Thr Ser Gln Phe Ala Ser Phe Ser Arg Asn Gln Ala Phe Thr 185 190 195 ggg aag caa ggc ggt gta gtt tac gct aca gga act ata act atc gag 739 Gly Lys Gln Gly Gly Val Val Tyr Ala Thr Gly Thr Ile Thr Ile Glu 200 205 210 aac agc cct ggg ata gtt tcc ttc tct caa aac cta gcg aaa gga tct 787 Asn Ser Pro Gly Ile Val Ser Phe Ser Gln Asn Leu Ala Lys Gly Ser 215 220 225 ggc ggt gct ctg tac agc act gac aac tgt tcg att aca gat aac ttt 835 Gly Gly Ala Leu Tyr Ser Thr Asp Asn Cys Ser Ile Thr Asp Asn Phe 230 235 240 245 caa gtg atc ttt gac ggc aat agt gct tgg gaa gcc gct caa gct cag 883 Gln Val Ile Phe Asp Gly Asn Ser Ala Trp Glu Ala Ala Gln Ala Gln 250 255 260 ggc ggg gct att tgt tgc act acg aca gat aaa aca gtg act ctt act 931 Gly Gly Ala Ile Cys Cys Thr Thr Thr Asp Lys Thr Val Thr Leu Thr 265 270 275 ggg aac aaa aac ctc tct ttc aca aat aat aca gca ttg aca tat ggc 979 Gly Asn Lys Asn Leu Ser Phe Thr Asn Asn Thr Ala Leu Thr Tyr Gly 280 285 290 gga gcc atc tct gga ctc aag gtc agt att tcc gct gga ggt cct act 1027 Gly Ala Ile Ser Gly Leu Lys Val Ser Ile Ser Ala Gly Gly Pro Thr 295 300 305 cta ttt caa agt aat atc tca gga agt agc gcc ggt cag gga gga gga 1075 Leu Phe Gln Ser Asn Ile Ser Gly Ser Ser Ala Gly Gln Gly Gly Gly 310 315 320 325 gga gcg atc aat ata gca tct gct ggg gaa ctc gct ctc tct gct act 1123 Gly Ala Ile Asn Ile Ala Ser Ala Gly Glu Leu Ala Leu Ser Ala Thr 330 335 340 tct gga gat att acc ttc aat aac aac caa gtc acc aac gga agc aca 1171 Ser Gly Asp Ile Thr Phe Asn Asn Asn Gln Val Thr Asn Gly Ser Thr 345 350 355 agt aca aga aac gca ata aat atc att gat acc gct aaa gtc aca tcg 1219 Ser Thr Arg Asn Ala Ile Asn Ile Ile Asp Thr Ala Lys Val Thr Ser 360 365 370 ata cga gct gct acg ggg caa tct atc tat ttc tat gat ccc atc aca 1267 Ile Arg Ala Ala Thr Gly Gln Ser Ile Tyr Phe Tyr Asp Pro Ile Thr 375 380 385 aat cca gga acc gca gct tct acc gac aca ttg aac tta aac tta gca 1315 Asn Pro Gly Thr Ala Ala Ser Thr Asp Thr Leu Asn Leu Asn Leu Ala 390 395 400 405 gat gcg aac agt gag atc gag tat ggg ggt gcg att gtc ttt tct gga 1363 Asp Ala Asn Ser Glu Ile Glu Tyr Gly Gly Ala Ile Val Phe Ser Gly 410 415 420 gaa aag ctt tcc cct aca gaa aaa gca atc gct gca aac gtc acc tct 1411 Glu Lys Leu Ser Pro Thr Glu Lys Ala Ile Ala Ala Asn Val Thr Ser 425 430 435 act atc cga caa cct gca gta tta gcg cgg gga gat ctt gta ctt cgt 1459 Thr Ile Arg Gln Pro Ala Val Leu Ala Arg Gly Asp Leu Val Leu Arg 440 445 450 gat gga gtc acc gta act ttc aag gat ctg act caa agt cca gga tcc 1507 Asp Gly Val Thr Val Thr Phe Lys Asp Leu Thr Gln Ser Pro Gly Ser 455 460 465 cgc atc tta atg gat ggg ggg act aca ctt agt gct aaa gag gca aat 1555 Arg Ile Leu Met Asp Gly Gly Thr Thr Leu Ser Ala Lys Glu Ala Asn 470 475 480 485 ctt tcg ctt aat ggc tta gca gta aat ctc tcc tct tta gat gga acc 1603 Leu Ser Leu Asn Gly Leu Ala Val Asn Leu Ser Ser Leu Asp Gly Thr 490 495 500 aac aag gca gct tta aaa aca gaa gct gca gat aaa aat atc agc cta 1651 Asn Lys Ala Ala Leu Lys Thr Glu Ala Ala Asp Lys Asn Ile Ser Leu 505 510 515 tcg gga acg att gcg ctt att gac acg gaa ggg tca ttc tat gag aat 1699 Ser Gly Thr Ile Ala Leu Ile Asp Thr Glu Gly Ser Phe Tyr Glu Asn 520 525 530 cat aac tta aaa agt gct agt acc tat cct ctt ctt gaa ctt acc acc 1747 His Asn Leu Lys Ser Ala Ser Thr Tyr Pro Leu Leu Glu Leu Thr Thr 535 540 545 gca gga gcc aac gga acg att act ctg gga gct ctt tct acc ctg act 1795 Ala Gly Ala Asn Gly Thr Ile Thr Leu Gly Ala Leu Ser Thr Leu Thr 550 555 560 565 ctt caa gaa cct gaa acc cac tac ggg tat caa gga aac tgg cag ttg 1843 Leu Gln Glu Pro Glu Thr His Tyr Gly Tyr Gln Gly Asn Trp Gln Leu 570 575 580 tct tgg gca aat gca aca tcc tca aaa ata gga agc atc aac tgg acc 1891 Ser Trp Ala Asn Ala Thr Ser Ser Lys Ile Gly Ser Ile Asn Trp Thr 585 590 595 cgt aca gga tac att cct agt cct gag aga aaa agt aat ctc cct cta 1939 Arg Thr Gly Tyr Ile Pro Ser Pro Glu Arg Lys Ser Asn Leu Pro Leu 600 605 610 aat agc tta tgg gga aac ttt ata gat ata cgc tcg atc aat cag ctt 1987 Asn Ser Leu Trp Gly Asn Phe Ile Asp Ile Arg Ser Ile Asn Gln Leu 615 620 625 ata gaa acc aag tcc agt ggg gag cct ttt gag cgt gag cta tgg ctt 2035 Ile Glu Thr Lys Ser Ser Gly Glu Pro Phe Glu Arg Glu Leu Trp Leu 630 635 640 645 tca gga att gcg aat ttc ttc tat aga gat tct atg ccc acc cgc cat 2083 Ser Gly Ile Ala Asn Phe Phe Tyr Arg Asp Ser Met Pro Thr Arg His 650 655 660 ggt ttc cgc cat atc agc ggg ggt tat gca cta ggg atc aca gca aca 2131 Gly Phe Arg His Ile Ser Gly Gly Tyr Ala Leu Gly Ile Thr Ala Thr 665 670 675 act cct gcc gag gat cag ctt act ttt gcc ttc tgc cag ctc ttt gct 2179 Thr Pro Ala Glu Asp Gln Leu Thr Phe Ala Phe Cys Gln Leu Phe Ala 680 685 690 aga gat cgc aat cat att aca ggt aag aac cac gga gat act tac ggt 2227 Arg Asp Arg Asn His Ile Thr Gly Lys Asn His Gly Asp Thr Tyr Gly 695 700 705 gcc tct ttg tat ttc cac cat aca gaa ggg ctc ttc gac atc gcc aat 2275 Ala Ser Leu Tyr Phe His His Thr Glu Gly Leu Phe Asp Ile Ala Asn 710 715 720 725 ttc ctc tgg gga aaa gca acc cga gct ccc tgg gtg ctc tct gag atc 2323 Phe Leu Trp Gly Lys Ala Thr Arg Ala Pro Trp Val Leu Ser Glu Ile 730 735 740 tcc cag atc att cct tta tcg ttc gat gct aaa ttc agt tat ctc cat 2371 Ser Gln Ile Ile Pro Leu Ser Phe Asp Ala Lys Phe Ser Tyr Leu His 745 750 755 aca gac aac cac atg aag aca tat tat acc gat aac tct atc atc aag 2419 Thr Asp Asn His Met Lys Thr Tyr Tyr Thr Asp Asn Ser Ile Ile Lys 760 765 770 ggt tct tgg aga aac gat gcc ttc tgt gca gat ctt gga gct agc ctg 2467 Gly Ser Trp Arg Asn Asp Ala Phe Cys Ala Asp Leu Gly Ala Ser Leu 775 780 785 cct ttt gtt att tcc gtt ccg tat ctt ctg aaa gaa gtc gaa cct ttt 2515 Pro Phe Val Ile Ser Val Pro Tyr Leu Leu Lys Glu Val Glu Pro Phe 790 795 800 805 gtc aaa gta cag tat atc tat gcg cat cag caa gac ttc tac gag cgt 2563 Val Lys Val Gln Tyr Ile Tyr Ala His Gln Gln Asp Phe Tyr Glu Arg 810 815 820 cat gct gaa gga cgc gct ttc aat aaa agc gag ctt atc aac gta gag 2611 His Ala Glu Gly Arg Ala Phe Asn Lys Ser Glu Leu Ile Asn Val Glu 825 830 835 att cct ata ggc gtc acc ttc gaa aga gac tca aaa tca gaa aag gga 2659 Ile Pro Ile Gly Val Thr Phe Glu Arg Asp Ser Lys Ser Glu Lys Gly 840 845 850 act tac gat ctt act ctt atg tat ata ctc gat gct tac cga cgc aat 2707 Thr Tyr Asp Leu Thr Leu Met Tyr Ile Leu Asp Ala Tyr Arg Arg Asn 855 860 865 cct aaa tgt caa act tcc cta ata gct agc gat gct aac tgg atg gcc 2755 Pro Lys Cys Gln Thr Ser Leu Ile Ala Ser Asp Ala Asn Trp Met Ala 870 875 880 885 tat ggt acc aac ctc gca cga caa ggt ttt tct gtt cgt gct gcg aac 2803 Tyr Gly Thr Asn Leu Ala Arg Gln Gly Phe Ser Val Arg Ala Ala Asn 890 895 900 cat ttc caa gtg aac ccc cac atg gaa atc ttc ggt caa ttc gct ttt 2851 His Phe Gln Val Asn Pro His Met Glu Ile Phe Gly Gln Phe Ala Phe 905 910 915 gaa gta cga agt tct tca cga aat tat aat aca aac cta ggc tct aag 2899 Glu Val Arg Ser Ser Ser Arg Asn Tyr Asn Thr Asn Leu Gly Ser Lys 920 925 930 ttt tgt ttc tagattatcg aaaacgtgtt aattaattga acccaagcat 2948 Phe Cys Phe 935 ctttctatga aaataccctt gcacaaactc ctgatctctt cgactcttgt cactcccatt 3008 ctattgagca ttgcaactta cggagcagat gcttctttat cc 3050 2 2808 DNA Chlamydia pneumoniae CDS (1)..(2808) 2 atgaagtcct ctgtctcttg gttgttcttt tcttcaatcc cgctcttttc atcgctctct 60 atagtcgcgg cagaggtgac cttagatagc agcaataata gctatgatgg atctaacgga 120 actaccttca cggtcttttc cactacggac gctgctgcag gaactaccta ttccttactt 180 tccgacgtat cctttcaaaa tgcaggggct ttaggaattc ccttagcctc aggatgcttc 240 ctagaagcgg gcggcgatct tactttccaa ggaaatcaac atgcactgaa gtttgcattt 300 atcaatgcgg gctctagcgc tggaactgta gccagtacct cagcagcaga taagaatctt 360 ctctttaatg atttttctag actctctatt atctcttgtc cctctcttct tctctctcct 420 actggacaat gtgctttaaa atctgtgggg aatctatctc taactggcaa ttcccaaatt 480 atatttactc agaacttctc gtcagataac ggcggtgtta tcaatacgaa aaacttctta 540 ttatcaggga catctcagtt tgcgagcttt tcgagaaacc aagccttcac agggaagcaa 600 ggcggtgtag tttacgctac aggaactata actatcgaga acagccctgg gatagtttcc 660 ttctctcaaa acctagcgaa aggatctggc ggtgctctgt acagcactga caactgttcg 720 attacagata actttcaagt gatctttgac ggcaatagtg cttgggaagc cgctcaagct 780 cagggcgggg ctatttgttg cactacgaca gataaaacag tgactcttac tgggaacaaa 840 aacctctctt tcacaaataa tacagcattg acatatggcg gagccatctc tggactcaag 900 gtcagtattt ccgctggagg tcctactcta tttcaaagta atatctcagg aagtagcgcc 960 ggtcagggag gaggaggagc gatcaatata gcatctgctg gggaactcgc tctctctgct 1020 acttctggag atattacctt caataacaac caagtcacca acggaagcac aagtacaaga 1080 aacgcaataa atatcattga taccgctaaa gtcacatcga tacgagctgc tacggggcaa 1140 tctatctatt tctatgatcc cattcacaaa tccaggaacc gcagcttcta ccgacacatt 1200 gaacttaaac ttagcagatg cgaacagtga gatcgagtat gggggtgcga ttgtcttttc 1260 tggagaaaag ctttccccta cagaaaaagc aatcgctgca aacgtcacct ctactatccg 1320 acaacctgca gtattagcgc ggggagatct tgtacttcgt gatggagtca ccgtaacttt 1380 caaggatctg actcaaagtc caggatcccg catcttaatg gatgggaggg atacacttag 1440 tgctaaagag gcaaatcttt cgcttaatgg cttagcagta aatctctcct ctttagatgg 1500 aaccaacaag gcagctttaa aaacagaagc tgcagataaa aatatcagcc tatcgggaac 1560 gattgcgctt attgacacgg aagggtcatt ctatgagaat cataacttaa aaagtgctag 1620 tacctatcct cttcttgaac ttaccaccgc aggagccaac ggaacgatta ctctgggagc 1680 tctttctacc ctgactcttc aagaacctga aacccactac gggtacaagg aaactggcag 1740 ttgtcttggg caaatgcaac atcctcaaaa ataggaagca tcaactggac ccgtacagga 1800 tacattccta gtcctgagag aaaaagtaat ctccctctaa atagcttatg gggaaacttt 1860 atagatatac gctcgatcaa tcagcttata gaaaccaagt ccagtgggga gccttttgag 1920 cgtgagctat ggctttcagg aattgcgaat ttcttctata gagattctat gcccacccgc 1980 catggtttcc gccatatcag cgggggttat gcactaggga tcacagcaac aactcctgcc 2040 gaggatcagc ttacttttgc cttctgccag ctctttgcta gagatcgcaa tcatattaca 2100 ggtaagaacc acggagatac ttacggtgcc tctttgtatt tccaccatac agaagggctc 2160 ttcgacatcg ccaatttcct ctggggaaaa gcaacccgag ctccctgggt gctctctgag 2220 atctcccaga tcattccttt atcgttcgat gctaaattca gttatctcca tacagacaac 2280 cacatgaaga catattatac cgataactct atcatcaagg gttcttggag aaacgatgcc 2340 ttctgtgcag atcttggagc tagcctgcct tttgttattt ccgttccgta acttctgaaa 2400 gaagtcgaac cttttgtcaa agtacagtat atctatgcgc atcagcaaga cttctacgag 2460 cgtcatgctg aaggacgcgc tttcaataaa agcgagctta tcaacgtaga gattcctata 2520 ggcgtcacct tcgaaagaga ctcaaaatca gaaaagggaa cttacgatct tactcttatg 2580 tatatactcg atgcttaccg acgcaatcct aaatgtcaaa cttccctaat agctagcgat 2640 gctaactgga tggcctatgg taccaacctc gcacgacaag gtttttctgt tcgtgctgcg 2700 aaccatttcc aagtgaaccc ccacatggaa atcttcggtc aattcgcttt tgaagtacga 2760 agttcttcac gaaattataa tacaaaccta ggctctaagt tttgtttc 2808 3 936 PRT Chlamydia pneumoniae 3 Met Lys Ser Ser Val Ser Trp Leu Phe Phe Ser Ser Ile Pro Leu Phe 1 5 10 15 Ser Ser Leu Ser Ile Val Ala Ala Glu Val Thr Leu Asp Ser Ser Asn 20 25 30 Asn Ser Tyr Asp Gly Ser Asn Gly Thr Thr Phe Thr Val Phe Ser Thr 35 40 45 Thr Asp Ala Ala Ala Gly Thr Thr Tyr Ser Leu Leu Ser Asp Val Ser 50 55 60 Phe Gln Asn Ala Gly Ala Leu Gly Ile Pro Leu Ala Ser Gly Cys Phe 65 70 75 80 Leu Glu Ala Gly Gly Asp Leu Thr Phe Gln Gly Asn Gln His Ala Leu 85 90 95 Lys Phe Ala Phe Ile Asn Ala Gly Ser Ser Ala Gly Thr Val Ala Ser 100 105 110 Thr Ser Ala Ala Asp Lys Asn Leu Leu Phe Asn Asp Phe Ser Arg Leu 115 120 125 Ser Ile Ile Ser Cys Pro Ser Leu Leu Leu Ser Pro Thr Gly Gln Cys 130 135 140 Ala Leu Lys Ser Val Gly Asn Leu Ser Leu Thr Gly Asn Ser Gln Ile 145 150 155 160 Ile Phe Thr Gln Asn Phe Ser Ser Asp Asn Gly Gly Val Ile Asn Thr 165 170 175 Lys Asn Phe Leu Leu Ser Gly Thr Ser Gln Phe Ala Ser Phe Ser Arg 180 185 190 Asn Gln Ala Phe Thr Gly Lys Gln Gly Gly Val Val Tyr Ala Thr Gly 195 200 205 Thr Ile Thr Ile Glu Asn Ser Pro Gly Ile Val Ser Phe Ser Gln Asn 210 215 220 Leu Ala Lys Gly Ser Gly Gly Ala Leu Tyr Ser Thr Asp Asn Cys Ser 225 230 235 240 Ile Thr Asp Asn Phe Gln Val Ile Phe Asp Gly Asn Ser Ala Trp Glu 245 250 255 Ala Ala Gln Ala Gln Gly Gly Ala Ile Cys Cys Thr Thr Thr Asp Lys 260 265 270 Thr Val Thr Leu Thr Gly Asn Lys Asn Leu Ser Phe Thr Asn Asn Thr 275 280 285 Ala Leu Thr Tyr Gly Gly Ala Ile Ser Gly Leu Lys Val Ser Ile Ser 290 295 300 Ala Gly Gly Pro Thr Leu Phe Gln Ser Asn Ile Ser Gly Ser Ser Ala 305 310 315 320 Gly Gln Gly Gly Gly Gly Ala Ile Asn Ile Ala Ser Ala Gly Glu Leu 325 330 335 Ala Leu Ser Ala Thr Ser Gly Asp Ile Thr Phe Asn Asn Asn Gln Val 340 345 350 Thr Asn Gly Ser Thr Ser Thr Arg Asn Ala Ile Asn Ile Ile Asp Thr 355 360 365 Ala Lys Val Thr Ser Ile Arg Ala Ala Thr Gly Gln Ser Ile Tyr Phe 370 375 380 Tyr Asp Pro Ile Thr Asn Pro Gly Thr Ala Ala Ser Thr Asp Thr Leu 385 390 395 400 Asn Leu Asn Leu Ala Asp Ala Asn Ser Glu Ile Glu Tyr Gly Gly Ala 405 410 415 Ile Val Phe Ser Gly Glu Lys Leu Ser Pro Thr Glu Lys Ala Ile Ala 420 425 430 Ala Asn Val Thr Ser Thr Ile Arg Gln Pro Ala Val Leu Ala Arg Gly 435 440 445 Asp Leu Val Leu Arg Asp Gly Val Thr Val Thr Phe Lys Asp Leu Thr 450 455 460 Gln Ser Pro Gly Ser Arg Ile Leu Met Asp Gly Gly Thr Thr Leu Ser 465 470 475 480 Ala Lys Glu Ala Asn Leu Ser Leu Asn Gly Leu Ala Val Asn Leu Ser 485 490 495 Ser Leu Asp Gly Thr Asn Lys Ala Ala Leu Lys Thr Glu Ala Ala Asp 500 505 510 Lys Asn Ile Ser Leu Ser Gly Thr Ile Ala Leu Ile Asp Thr Glu Gly 515 520 525 Ser Phe Tyr Glu Asn His Asn Leu Lys Ser Ala Ser Thr Tyr Pro Leu 530 535 540 Leu Glu Leu Thr Thr Ala Gly Ala Asn Gly Thr Ile Thr Leu Gly Ala 545 550 555 560 Leu Ser Thr Leu Thr Leu Gln Glu Pro Glu Thr His Tyr Gly Tyr Gln 565 570 575 Gly Asn Trp Gln Leu Ser Trp Ala Asn Ala Thr Ser Ser Lys Ile Gly 580 585 590 Ser Ile Asn Trp Thr Arg Thr Gly Tyr Ile Pro Ser Pro Glu Arg Lys 595 600 605 Ser Asn Leu Pro Leu Asn Ser Leu Trp Gly Asn Phe Ile Asp Ile Arg 610 615 620 Ser Ile Asn Gln Leu Ile Glu Thr Lys Ser Ser Gly Glu Pro Phe Glu 625 630 635 640 Arg Glu Leu Trp Leu Ser Gly Ile Ala Asn Phe Phe Tyr Arg Asp Ser 645 650 655 Met Pro Thr Arg His Gly Phe Arg His Ile Ser Gly Gly Tyr Ala Leu 660 665 670 Gly Ile Thr Ala Thr Thr Pro Ala Glu Asp Gln Leu Thr Phe Ala Phe 675 680 685 Cys Gln Leu Phe Ala Arg Asp Arg Asn His Ile Thr Gly Lys Asn His 690 695 700 Gly Asp Thr Tyr Gly Ala Ser Leu Tyr Phe His His Thr Glu Gly Leu 705 710 715 720 Phe Asp Ile Ala Asn Phe Leu Trp Gly Lys Ala Thr Arg Ala Pro Trp 725 730 735 Val Leu Ser Glu Ile Ser Gln Ile Ile Pro Leu Ser Phe Asp Ala Lys 740 745 750 Phe Ser Tyr Leu His Thr Asp Asn His Met Lys Thr Tyr Tyr Thr Asp 755 760 765 Asn Ser Ile Ile Lys Gly Ser Trp Arg Asn Asp Ala Phe Cys Ala Asp 770 775 780 Leu Gly Ala Ser Leu Pro Phe Val Ile Ser Val Pro Tyr Leu Leu Lys 785 790 795 800 Glu Val Glu Pro Phe Val Lys Val Gln Tyr Ile Tyr Ala His Gln Gln 805 810 815 Asp Phe Tyr Glu Arg His Ala Glu Gly Arg Ala Phe Asn Lys Ser Glu 820 825 830 Leu Ile Asn Val Glu Ile Pro Ile Gly Val Thr Phe Glu Arg Asp Ser 835 840 845 Lys Ser Glu Lys Gly Thr Tyr Asp Leu Thr Leu Met Tyr Ile Leu Asp 850 855 860 Ala Tyr Arg Arg Asn Pro Lys Cys Gln Thr Ser Leu Ile Ala Ser Asp 865 870 875 880 Ala Asn Trp Met Ala Tyr Gly Thr Asn Leu Ala Arg Gln Gly Phe Ser 885 890 895 Val Arg Ala Ala Asn His Phe Gln Val Asn Pro His Met Glu Ile Phe 900 905 910 Gly Gln Phe Ala Phe Glu Val Arg Ser Ser Ser Arg Asn Tyr Asn Thr 915 920 925 Asn Leu Gly Ser Lys Phe Cys Phe 930 935 4 925 PRT Chlamydia pneumoniae 4 Ser Ile Pro Leu Phe Ser Ser Leu Ser Ile Val Ala Ala Glu Val Thr 1 5 10 15 Leu Asp Ser Ser Asn Asn Ser Tyr Asp Gly Ser Asn Gly Thr Thr Phe 20 25 30 Thr Val Phe Ser Thr Thr Asp Ala Ala Ala Gly Thr Thr Tyr Ser Leu 35 40 45 Leu Ser Asp Val Ser Phe Gln Asn Ala Gly Ala Leu Gly Ile Pro Leu 50 55 60 Ala Ser Gly Cys Phe Leu Glu Ala Gly Gly Asp Leu Thr Phe Gln Gly 65 70 75 80 Asn Gln His Ala Leu Lys Phe Ala Phe Ile Asn Ala Gly Ser Ser Ala 85 90 95 Gly Thr Val Ala Ser Thr Ser Ala Ala Asp Lys Asn Leu Leu Phe Asn 100 105 110 Asp Phe Ser Arg Leu Ser Ile Ile Ser Cys Pro Ser Leu Leu Leu Ser 115 120 125 Pro Thr Gly Gln Cys Ala Leu Lys Ser Val Gly Asn Leu Ser Leu Thr 130 135 140 Gly Asn Ser Gln Ile Ile Phe Thr Gln Asn Phe Ser Ser Asp Asn Gly 145 150 155 160 Gly Val Ile Asn Thr Lys Asn Phe Leu Leu Ser Gly Thr Ser Gln Phe 165 170 175 Ala Ser Phe Ser Arg Asn Gln Ala Phe Thr Gly Lys Gln Gly Gly Val 180 185 190 Val Tyr Ala Thr Gly Thr Ile Thr Ile Glu Asn Ser Pro Gly Ile Val 195 200 205 Ser Phe Ser Gln Asn Leu Ala Lys Gly Ser Gly Gly Ala Leu Tyr Ser 210 215 220 Thr Asp Asn Cys Ser Ile Thr Asp Asn Phe Gln Val Ile Phe Asp Gly 225 230 235 240 Asn Ser Ala Trp Glu Ala Ala Gln Ala Gln Gly Gly Ala Ile Cys Cys 245 250 255 Thr Thr Thr Asp Lys Thr Val Thr Leu Thr Gly Asn Lys Asn Leu Ser 260 265 270 Phe Thr Asn Asn Thr Ala Leu Thr Tyr Gly Gly Ala Ile Ser Gly Leu 275 280 285 Lys Val Ser Ile Ser Ala Gly Gly Pro Thr Leu Phe Gln Ser Asn Ile 290 295 300 Ser Gly Ser Ser Ala Gly Gln Gly Gly Gly Gly Ala Ile Asn Ile Ala 305 310 315 320 Ser Ala Gly Glu Leu Ala Leu Ser Ala Thr Ser Gly Asp Ile Thr Phe 325 330 335 Asn Asn Asn Gln Val Thr Asn Gly Ser Thr Ser Thr Arg Asn Ala Ile 340 345 350 Asn Ile Ile Asp Thr Ala Lys Val Thr Ser Ile Arg Ala Ala Thr Gly 355 360 365 Gln Ser Ile Tyr Phe Tyr Asp Pro Ile Thr Asn Pro Gly Thr Ala Ala 370 375 380 Ser Thr Asp Thr Leu Asn Leu Asn Leu Ala Asp Ala Asn Ser Glu Ile 385 390 395 400 Glu Tyr Gly Gly Ala Ile Val Phe Ser Gly Glu Lys Leu Ser Pro Thr 405 410 415 Glu Lys Ala Ile Ala Ala Asn Val Thr Ser Thr Ile Arg Gln Pro Ala 420 425 430 Val Leu Ala Arg Gly Asp Leu Val Leu Arg Asp Gly Val Thr Val Thr 435 440 445 Phe Lys Asp Leu Thr Gln Ser Pro Gly Ser Arg Ile Leu Met Asp Gly 450 455 460 Gly Thr Thr Leu Ser Ala Lys Glu Ala Asn Leu Ser Leu Asn Gly Leu 465 470 475 480 Ala Val Asn Leu Ser Ser Leu Asp Gly Thr Asn Lys Ala Ala Leu Lys 485 490 495 Thr Glu Ala Ala Asp Lys Asn Ile Ser Leu Ser Gly Thr Ile Ala Leu 500 505 510 Ile Asp Thr Glu Gly Ser Phe Tyr Glu Asn His Asn Leu Lys Ser Ala 515 520 525 Ser Thr Tyr Pro Leu Leu Glu Leu Thr Thr Ala Gly Ala Asn Gly Thr 530 535 540 Ile Thr Leu Gly Ala Leu Ser Thr Leu Thr Leu Gln Glu Pro Glu Thr 545 550 555 560 His Tyr Gly Tyr Gln Gly Asn Trp Gln Leu Ser Trp Ala Asn Ala Thr 565 570 575 Ser Ser Lys Ile Gly Ser Ile Asn Trp Thr Arg Thr Gly Tyr Ile Pro 580 585 590 Ser Pro Glu Arg Lys Ser Asn Leu Pro Leu Asn Ser Leu Trp Gly Asn 595 600 605 Phe Ile Asp Ile Arg Ser Ile Asn Gln Leu Ile Glu Thr Lys Ser Ser 610 615 620 Gly Glu Pro Phe Glu Arg Glu Leu Trp Leu Ser Gly Ile Ala Asn Phe 625 630 635 640 Phe Tyr Arg Asp Ser Met Pro Thr Arg His Gly Phe Arg His Ile Ser 645 650 655 Gly Gly Tyr Ala Leu Gly Ile Thr Ala Thr Thr Pro Ala Glu Asp Gln 660 665 670 Leu Thr Phe Ala Phe Cys Gln Leu Phe Ala Arg Asp Arg Asn His Ile 675 680 685 Thr Gly Lys Asn His Gly Asp Thr Tyr Gly Ala Ser Leu Tyr Phe His 690 695 700 His Thr Glu Gly Leu Phe Asp Ile Ala Asn Phe Leu Trp Gly Lys Ala 705 710 715 720 Thr Arg Ala Pro Trp Val Leu Ser Glu Ile Ser Gln Ile Ile Pro Leu 725 730 735 Ser Phe Asp Ala Lys Phe Ser Tyr Leu His Thr Asp Asn His Met Lys 740 745 750 Thr Tyr Tyr Thr Asp Asn Ser Ile Ile Lys Gly Ser Trp Arg Asn Asp 755 760 765 Ala Phe Cys Ala Asp Leu Gly Ala Ser Leu Pro Phe Val Ile Ser Val 770 775 780 Pro Tyr Leu Leu Lys Glu Val Glu Pro Phe Val Lys Val Gln Tyr Ile 785 790 795 800 Tyr Ala His Gln Gln Asp Phe Tyr Glu Arg His Ala Glu Gly Arg Ala 805 810 815 Phe Asn Lys Ser Glu Leu Ile Asn Val Glu Ile Pro Ile Gly Val Thr 820 825 830 Phe Glu Arg Asp Ser Lys Ser Glu Lys Gly Thr Tyr Asp Leu Thr Leu 835 840 845 Met Tyr Ile Leu Asp Ala Tyr Arg Arg Asn Pro Lys Cys Gln Thr Ser 850 855 860 Leu Ile Ala Ser Asp Ala Asn Trp Met Ala Tyr Gly Thr Asn Leu Ala 865 870 875 880 Arg Gln Gly Phe Ser Val Arg Ala Ala Asn His Phe Gln Val Asn Pro 885 890 895 His Met Glu Ile Phe Gly Gln Phe Ala Phe Glu Val Arg Ser Ser Ser 900 905 910 Arg Asn Tyr Asn Thr Asn Leu Gly Ser Lys Phe Cys Phe 915 920 925 5 43 DNA Artificial Sequence primer (5′) 5 ataagaatgc ggccgccacc atggcagagg tgaccttaga tag 43 6 31 DNA Artificial Sequence primer (3′) 6 cggctcgagt gaaacaaaac ttagagccta g 31 

1. A nucleic acid molecule comprising a nucleic acid sequence which encodes a polypeptide selected from any of: (a) SEQ ID Nos: 3 and 4; (b) an immunogenic fragment comprising at least 12 consecutive amino acids from a polypeptide of (a); and (c) a polypeptide of (a) or (b) which has been modified to improve its immunogenicity, wherein said modified polypeptide is at least 75% identical in amino acid sequence to the corresponding polypeptide of (a) or (b).
 2. A nucleic acid molecule comprising a nucleic acid sequence selected from any of: (a) SEQ ID Nos: 1 and 2; (b) a sequence which encodes a polypeptide encoded by SEQ ID No: 1 or 2; (c) a sequence comprising at least 38 consecutive nucleotides from any one of the nucleic acid sequences of (a) and (b); and (d) a sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptides encoded by SEQ ID No: 1 or
 2. 3. A nucleic acid molecule comprising a nucleic acid sequence which is anti-sense to the nucleic acid molecule of claim 1 or
 2. 4. A nucleic acid molecule comprising a nucleic acid sequence which encodes a fusion protein, said fusion protein comprising a polypeptide encoded by a nucleic acid molecule according to claim 1 and an additional polypeptide.
 5. The nucleic acid molecule of claim 4 wherein the additional polypeptide is a heterologous signal peptide.
 6. The nucleic acid molecule of claim 4 wherein the additional polypeptide has adjuvant activity.
 7. A nucleic acid molecule according to any one of claims 1 to 6, operatively linked to one or more expression control sequences.
 8. A vaccine comprising at least one first nucleic acid according to any one of claims 1, 2, and 4 to 7 and a vaccine vector wherein each first nucleic acid is expressed as a polypeptide, the vaccine optionally comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by said first nucleic acid.
 9. The vaccine of claim 8 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
 10. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.
 11. A pharmaceutical composition comprising a vaccine according to claim 8 or 9 and a pharmaceutically acceptable carrier.
 12. A unicellular host transformed with the nucleic acid molecule of claim
 7. 13. A nucleic acid probe of 5 to 100 nucleotides which hybridizes under stringent conditions to the nucleic acid molecule of SEQ ID No: 1 or 2, or to a homolog or complementary or anti-sense sequence of said nucleic acid molecule.
 14. A primer of 10 to 40 nucleotides which hybridizes under stringent conditions to the nucleic acid molecules of SEQ ID No: 1 or 2, or to a homolog or complementary or anti-sense sequence of said nucleic acid molecule.
 15. A polypeptide encoded by a nucleic acid sequence according to any one of claims 1, 2 and 4 to
 7. 16. A polypeptide comprising an amino acid sequence selected from any of: (a) SEQ ID Nos: 3 and 4; (b) an immunogenic fragment comprising at least 12 consecutive amino acids from a polypeptide of (a); and (c) a polypeptide of (a) or (b) which has been modified to improve its immunogenicity, wherein said modified polypeptide is at least 75% identical in amino acid sequence to the corresponding polypeptide of (a) or (b).
 17. A fusion polypeptide comprising a polypeptide of claim 15 or 16 and an additional polypeptide.
 18. The fusion polypeptide of claim 17 wherein the additional polypeptide is a heterologous signal peptide.
 19. The fusion protein of claim 17 wherein the additional polypeptide has adjuvant activity.
 20. A method for producing a polypeptide of claim 15 or 16, comprising the step of culturing a unicellular host according to claim
 12. 21. An antibody against the polypeptide of any one of claims 15 to
 19. 22. A vaccine comprising at least one first polypeptide according to any one of claims 15 to 19 and a pharmaceutically acceptable carrier, optionally comprising a second polypeptide which enhances the immune response to the first polypeptide.
 23. The vaccine of claim 22 wherein the second polypeptide comprises an additional Chlamydia polypeptide.
 24. A pharmaceutical composition comprising a polypeptide according to any one of claims 15 to 19 and a pharmaceutically acceptable carrier.
 25. A pharmaceutical composition comprising a vaccine according to claim 22 or 23 and a pharmaceutically acceptable carrier.
 26. A pharmaceutical composition comprising an antibody according to claim 21 and a pharmaceutically acceptable carrier.
 27. A method for preventing or treating Chlamydia infection using: (a) the nucleic acid of any one of claims 1 to 7; (b) the vaccine of any one of claims 8, 9, 22 and 23; (c) the pharmaceutical composition of any one of claims 10, 11, 24 to 26; (d) the polypeptide of any one of claims 15 to 19; or (e) the antibody of claim
 21. 28. A method of detecting Chiamydia infection comprising he step of assaying a body fluid of a mammal to be tested, with component selected from any one of: (a) the nucleic acid of any one of claims 1 to 7; (b) the polypeptide of any one of claims 15 to 19; and (c) the antibody of claim
 21. 29. A diagnostic kit comprising instructions for use and a component selected from any one of: (a) the nucleic acid of any one of claims 1 to 7; (b) the polypeptide of any one of claims 15 to 19; and (c) the antibody of claim
 21. 30. A method for identifying a polypeptide of claims 15 to 19 which induces an immune response effective to prevent or lessen the severity of Chlamydia infection in a mammal previously immunized with polypeptide, comprising the steps of: (a) immunizing a mouse with the polypeptide; and (b) inoculating the immunized mouse with Chlamydia; wherein the polypeptide which prevents or lessens the severity of Chlamydia infection in the immunized mouse compared to a non-immunized control mouse is identified.
 31. Expression plasmid pCAI640. 